System and method for monitoring cardiomyocyte beating, viability, morphology, and electrophysiological properties

ABSTRACT

Devices, systems and methods for monitoring excitable cells, such as cardiomyocytes, on microelectrode arrays that couple the electro-stimulation of excitable cells to induce or regulate cardiomyocyte beating and the simultaneous measurement of impedance and extracellular recording to assess changes in cardiomyocyte beating, viability, morphology or electrophysical properties in response to a plurality of treatments.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority to U.S. provisional patentapplication Ser. No. 61/730,427 filed Nov. 27, 2012, entitled, “Systemand method for monitoring cardiomyocyte beating, viability, morphologyand electrophysiological properties.”

This application is also a continuation in part of U.S. patentapplication Ser. No. 12/774,709 filed May 5, 2010, entitled, “System andmethod for monitoring cardiomyocyte beating, viability and morphologyand for screening for pharmacological agents which may inducecardiotoxicity or modulate cardiomyocyte function”, which is acontinuation in part of U.S. patent application Ser. No. 12/435,569,filed May 5, 2009, entitled, “Label free monitoring ofexcitation-contraction coupling and excitable cells using impedancebased systems with millisecond time resolution,” which claims priorityto U.S. provisional patent application Ser. No. 61/191,684, filed Sep.11, 2008, and U.S. provisional patent application Ser. No. 61/126,533,filed May 5, 2008; the contents of each are herein incorporated byreference in their entirety.

Parent U.S. patent application Ser. No. 12/774,709 also claims priorityto U.S. patent application Ser. No. 61/323,782, filed on Apr. 13, 2010,entitled, “Impedance based monitoring of cardiomyocytes”; U.S. patentapplication Ser. No. 61/310,557, filed Mar. 15, 2010, entitled,“Impedance based monitoring of cardiomyocytes”; and U.S. patentapplication Ser. No. 61/175,566, filed May 5, 2009, entitled, “Systemand method for monitoring cardiomyocyte beating, viability andmorphology and for screening for pharmacological agents which may inducecardiotoxicity or modulate cardiomyocyte function.” All of theapplications referred to in this paragraph are incorporated by referencein their entireties herein.

Parent U.S. patent application Ser. No. 12/774,709 is also acontinuation in part of U.S. patent application Ser. No. 11/235,938entitled, “Dynamic Monitoring of Cell Adhesion and Spreading Using theRT-CES System”, filed on Sep. 27, 2005, now U.S. Pat. No. 7,732,127,which is a continuation in part of U.S. patent application Ser. No.11/197,994, now U.S. Pat. No. 7,468,255, entitled, “Method for Assayingfor Natural Killer, Cytotoxic T-Lymphocyte and Neutrophil-MediatedKilling of Target Cells Using Real-

Parent U.S. patent application Ser. No. 12/774,709 is also acontinuation in part of U.S. patent application Ser. No. 11/235,938entitled, “Dynamic Monitoring of Cell Adhesion and Spreading Using theRT-CES System”, filed on Sep. 27, 2005, now U.S. Pat. No. 7,732,127,which is a continuation in part of U.S. patent application Ser. No.11/197,994, now U.S. Pat. No. 7,468,255, entitled, “Method for Assayingfor Natural Killer, Cytotoxic T-Lymphocyte and Neutrophil-MediatedKilling of Target Cells Using Real-Time Microelectronic Cell SensingTechnology”, filed on Aug. 4, 2005, which is a continuation-in-part ofU.S. patent application Ser. No. 11/055,639, now U.S. Pat. No.7,560,269, entitled “Real time electronic cell sensing system andapplications for cytotoxicity profiling and compound assays” filed onFeb. 9, 2005 which is a continuation-in-part of U.S. patent applicationSer. No. 10/987,732, now U.S. Pat. No. 7,192,752, entitled “Real timeelectronic cell sensing system and application for cell based assays”filed on Nov. 12, 2004, which claims priority to U.S. provisionalapplication Ser. No. 60/519,567, filed on Nov. 12, 2003. Allapplications referred to in this paragraph are incorporated by referencein their entireties herein.

Parent U.S. patent application Ser. No. 11/235,938, also claims benefitof priority to U.S. provisional patent application Ser. No. 60/630,131,filed on Nov. 22, 2004; U.S. provisional patent application Ser. No.60/630,071 filed on Nov. 22, 2004; U.S. provisional patent applicationSer. No. 60/613,872 filed on Sep. 27, 2004; U.S. provisional patentapplication Ser. No. 60/613,749, filed on Sep. 27, 2004; U.S.provisional patent application Ser. No. 60/630,809 filed on Nov. 24,2004; U.S. provisional patent application Ser. No. 60/633,019 filed onDec. 3, 2004; U.S. provisional patent application Ser. No. 60/647,159filed on Jan. 26, 2005; U.S. provisional patent application Ser. No.60/653,904 filed on Feb. 17, 2005; and U.S. provisional patentapplication Ser. No. 60/673,678 filed on Apr. 25, 2005; U.S. provisionalpatent application Ser. No. 60/689,422 filed on Jun. 10, 2005. All ofthe applications referred to in this paragraph are incorporated byreference in their entireties herein.

Parent U.S. patent application Ser. No. 11/235,938 is also acontinuation-in-part of U.S. patent application Ser. No. 11/198,831,entitled, “Dynamic Monitoring of Activation of G-Protein CoupledReceptor (GPCR) and Receptor Tyrosine Kinase (RTK) in Living Cells usingReal-Time Microelectronic Cell Sensing Technology, filed on Aug. 4,2005, now U.S. Pat. No. 8,263,375, which is herein incorporated byreference in its entirety.

Parent U.S. patent application Ser. No. 10/987,732, now U.S. Pat. No.7,192,752, is also a continuation-in-part of U.S. patent applicationSer. No. 10/705,615, now U.S. Pat. No. 7,459,303, entitled “ImpedanceBased Apparatuses and Methods for Analyzing Cells and Particles”, filedon Nov. 10, 2003, which claims priority to U.S. provisional patentapplication Ser. No. 60/397,749 filed on Jul. 20, 2002; U.S. provisionalpatent application Ser. 60/435,400, filed on Dec. 20, 2002; and U.S.provisional patent application Ser. No. 60/469,572, filed on May 9,2003. All of the applications referred to in this paragraph areincorporated by reference in their entireties herein.

Parent U.S. patent application Ser. No. 10/987,732, now U.S. Pat. No.7,192,752, is also a continuation in part of U.S. patent applicationSer. No. 10/705,447, now U.S. Pat. No. 7,470,533, filed on Nov. 10,2003, entitled “Impedance Based Devices and Methods for Use in Assays”which claims priority to U.S. provisional patent application Ser. No.60/397,749, filed on Jul. 20, 2002; U.S. provisional patent applicationSer. No. 60/435,400, filed on Dec. 20, 2002; and U.S. provisional patentapplication Ser. No. 60/469,572, filed on May 9, 2003. All of theapplications referred to in this paragraph are incorporated by referencein their entireties herein.

Parent U.S. patent application Ser. No. 11/055,639, now U.S. Pat. No.7,560,269 also claims priority to U.S. provisional patent applicationSer. No. 60/542,927 filed on Feb. 9, 2004; U.S. provisional patentapplication Ser. No. 60/548,713, filed on Feb. 27, 2004, and U.S.provisional patent application Ser. No. 60/614,601, filed on Sep. 29,2004. All of the applications referred to in this paragraph areincorporated by reference in their entireties herein.

Parent U.S. patent application Ser. No. 11/197,994, now U.S. Pat. No.7,468,255 also claims priority to U.S. provisional patent applicationSer. No. 60/598,608, filed on Aug. 4, 2004, U.S. provisional patentapplication Ser. No. 60/630,131, filed on Nov. 22, 2004, U.S.provisional patent application Ser. No. 60/689,422, filed on Jun. 10,2005, U.S. provisional patent application Ser. No. 60/598,609, filed onAug. 4, 2004, U.S. provisional patent application Ser. No. 60/613,749,filed on Sep. 27, 2004, U.S. provisional patent application Ser. No.60/647,189, filed on Jan. 26, 2005, U.S. provisional patent applicationSer. No. 60/647,075, filed on Jan. 26, 2005, U.S. provisional patentapplication Ser. No. 60/660,829, filed on Mar. 10, 2005, and U.S.provisional patent application Ser. No. 60/660,898, filed on Mar. 10,2005. All of the applications referred to in this paragraph areincorporated by reference in their entireties herein.

TECHNICAL FIELD

This invention relates to the field of cell-based assays and morespecifically to devices, systems and methods for electro-stimulation ofexcitable cells, such as cardiomyocyte or cardiomyocyte precursor cellsand performing extracellular recording and/or impedance monitoring ofelectro-stimulated cells.

BACKGROUND OF THE INVENTION

Bioelectronics is a progressing interdisciplinary research field thatinvolves the integration of biomaterials with electronic devices.Bioelectronics methods have been used for analyzing cells and assayingbiological molecules and cells. In one type of application, cells arecultured on microelectrodes and cell-electrode impedance orcell-substrate impedance is measured and analyzed to monitor cellularchanges, such as changes in cell morphology. For example, PCTApplication No. PCT/US03/22557, titled “Impedance Based Devices andMethods for Use in Assays”, discloses a device for detecting cellsand/or molecules on an electrode surface. The device detects cellsand/or molecules through measurement of impedance changes resulting fromthe attachment or binding of cells and/or molecules to the electrodesurfaces. A number of embodiments of the device are disclosed, togetherwith the apparatuses, and systems for using such devices to performcertain cell based assays.

Bringing a new drug to the market can take anywhere between 8 to 16years, and the average cost of developing a drug is now around $500-$800million with the cost expected to hit the $1 billion mark within thenext four years. Cardiotoxicity has been cited as the reason for 30percent of all failed drug compounds during development and is a majorcause of compound attrition. The late detection of cardiotoxic sideeffects caused by pharmacological compounds can impede drug discoveryand development projects, and consequently increase their cost. Testingfor the potential cardiotoxic side effects of compounds at an earlystage of drug development has therefore been the goal of manypharmaceutical and biotechnology companies. Cardiotoxicity itself canentail a number of short-term and long term cellular events includingdirectly affecting the beating rate of cardiomyocytes, viability ofcardiomyocytes and morphology of cardiomyocytes as would occur inhypertrophy. The core of the current issue in pharmacological safetyassessment and drug development is the lack of a reliable screeningmethodology capable of monitoring potential drug-mediated cardiotoxicityand distinguishing between different modes of cardiotoxicity. What isurgently needed in the field is a good cell-based model system as wellas a monitoring system with a physiological and functional readout thatcan provide incisive information regarding potential cardiotoxic sideeffects of drugs.

Traditionally, the drug discovery industry has undertaken two differentapproaches for toxicological assessment of drug candidate leads incardiac function. The first approach involves isolation ofcardiomyocytes directly from a mammalian species such as rats and dogsfollowed by electrophysiological and viability studies on the isolatedcardiomyocytes. This approach is extremely labor-intensive, timeconsuming and costly and at the same time not very amenable to the highthroughput demands of pharmaceutical industry

An alternative method for prediction of cardiotoxicity of drug candidateleads early in the drug development process has involved utilizingcell-based assay models which heterologously express specific ionchannels such as hERG channels or voltage-gated calcium channels. Thesecardiac ion channels have been envisioned as possible molecular targetsthrough which drugs could induce cardiotoxicity. These cell-basedsystems allow the assessment of drug-channel interaction by monitoringthe effect of the drug on the currents produced by the differentchannels in cultured cells using a technique known as ‘patch clamping’,which isolates regions of the cell membrane containing channel proteinsand measures changes in electrical potential difference. Use of thismethod in high throughput requires automation of patch clamping in arrayformat, which even though is available in last several years is not yetwidespread. Another issue with this approach is that cardiac toxicitymay occur by other mechanisms which can easily be missed by this type oftargeted approach.

An alternative to the in vitro ion-channel recording assays as well asthe labor-intensive isolation of primary tissue is to utilize thedifferentiation of embryonic stem (ES) cells into cardiomyocytes as astarting material for functional assays. The utility of ES cells as atreatment for various chronic diseases has received much attention inrecent years. Mammalian ES cells are self-renewing cells derived fromthe inner cell mass of a blastocyst stage embryo, which can bedifferentiated into multiple different cell types. It has beendemonstrated that the mouse ES cells as well as human ES cells can bedifferentiated into cardiomyocytes, which retain the ability to beat inculture.

The differentiation of ES cells first involves an intermediate in vitrodevelopmental stage in which ES cells form compact cell structures knownas embryoid bodies. These embryoid bodies can induce the developmentalprogram of ES cell differentiation into multiple cell types includingcardiomyocytes, which are distinguished in culture by their ability toundergo spontaneous beating. These ES derived in vitro differentiatedcardiomyocytes recapitulates the normal development of cardiomyocytes asevidenced by the stage-specific expression of cardiomyocyte specificgenes. All the known transcription factors, ion channels and structuralproteins that are part of normal heart development and function in vivoare also expressed in ES-derived cardiomyocyte.

Because ES cells are self-renewing, cells in culture can serve as anexcellent source for continuous production of cardiomyocytes. Therefore,these cardiomyocytes which behave in every way like normalcardiomyocytes isolated from the heart tissue itself addresses the everimportant supply problem and for the first time allows for assessment ofcardiac function and its modulation by lead candidate drugs andcompounds in relatively large scale in both viability assays, assessmentof morphology and in monitoring the beating function of cardiomyocytes.Furthermore, because the technology exists to selectively knockout orexpress trans-genes in ES cells, it provides an excellent model systemto study the role of certain genes in cardiac development and functionwithout having to be concerned about adverse affects on overallembryonic development in transgenic animals.

The ability to express transgenes in ES cells has been utilized as a wayto enrich for preparation of cardiomyocytes that are 100% pure. Forexample, the gene encoding GFP has been cloned downstream of acardiac-specific promoter and then introduced into ES cells. Embryoidcells, which ultimately differentiate into cardiomyocytes, willtherefore express the GFP transgenes and these cells can be easilyisolated by cell sorting techniques and therefore an enrichedcardiomyocyte population can be obtained.

Technologies designed to assess cardiomyocyte behavior and function andthe effect of drugs and other manipulations in vitro can be divided intotwo different approaches. One approach involves long term assessment ofcardiomyocyte viability for example in response to certain compounds.Such assays are typically end point assays designed to measure acellular component such as ATP, which correlates with the degree ofviability of the cells. The other approach involves studying short termeffect of drugs and compounds on beating function of cardiomyocytes.High throughput techniques for short term functional characterization ofion channels and other targets in cardiomyocytes has been ratherchallenging and limited. Systems such as automatic patch clampinstrumentation that are available can monitor a single cardiomyocyte ata time and with very limited throughput.

In US 2011/0039294 an approach to monitoring a cardiomyocyte populationis disclosed, which includes both impedance monitoring and extracellularrecording technologies with high precision. While acceptance of thetechnology is increasing, there still remain challenges when workingwith the cells themselves. For example, when using primary cardiomyocytecells harvested from tissue or after extended culturing, the beating ofcells can stop or slow. Accordingly, there is a need to provide devices,systems and methods that assess the cardiotoxicity of compounds whileworking to ensure the continued or regular beating of cardiomyocytecells.

SUMMARY OF THE INVENTION

The present invention discloses devices, systems and methods forperforming impedance monitoring and/or extracellular recording ofexcitable cells, such as cardiomyocytes in cell based assays thatencourage the continued and regular beating of excitable cells such thatthe cardiotoxicity of potential drugs can be assessed. In addition, thedevices, systems and methods provide improved characterization ofexcitable cells in response to compound administration to assessresponses of cardiomyocytes including cardiotoxic effects, whileincreasing efficiency of throughput for potential drug candidates. Theinvention further provides improved characterization of cells duringdifferentiation processes related to development of cardiomyocytes.

In one aspect of the invention a system for monitoring excitable cellsis disclosed, which includes a device having at least one well, eachwell having a bottom having a nonconductive substrate, wherein thesubstrate has a surface suitable for attachment of excitable cells; apower source configured to deliver an electrical signal capable ofelectro-stimulating excitable cells; and at least one analyzing modulefor measuring an electrical property from electro-stimulated excitablecells, characterized in that each well includes a pair ofelectro-stimulation electrodes configured to receive the electricalsignal from the power source thereby delivering an electro-stimulatingsignal to the well for electro-stimulation of excitable cells attachedto the substrate; and at least a second pair of electrodescommunicatively coupled to the at least one analyzing module, which isselected from the group consisting of a pair of impedance monitoringelectrodes communicatively coupled to the at least one analyzing modulein the form of an impedance analyzer thereby permitting impedancemonitoring of excitable cells attached to the substrate, and anextracellular recording electrode pair communicatively coupled to the atleast one analyzing module in the form of an extracellular recordingamplifier thereby permitting extracellular recording of excitable cellsattached to the substrate.

In regards to electro-stimulation, in some embodiments the electricalsignal is a series of pulses at a regular time interval, such as but notlimited to 0.5 seconds to 2 seconds. The electrical signal can vary butis preferably 1V to 2.5V for 0.5-2 milliseconds. A percentage of asurface area of the bottom of the at least one well occupied by the pairof electro-stimulation electrodes can vary but in some embodiments isselected from the group consisting of 5% or more, 10% or more, 20% ormore, 30% or more, 50% or more, and 70% or more. The electro-stimulationelectrodes independently includes an unbranched electrode structure or abranched electrode structure.

In preferred impedance monitoring configurations, the at least secondpair of electrodes is the pair of impedance monitoring electrodes andthe analyzing module is in the form of the impedance analyzer. The pairof impedance monitoring electrodes can be provided in a variety ofconfigurations but is preferably a pair of interdigitated electrodestructures, wherein each electrode structure comprises a plurality ofelectrode elements. In addition, the pair of impedance monitoringelectrodes can be a pair of electrode structures having a same surfacearea. In preferred embodiments the impedance analyzer monitors impedanceat millisecond time resolution In further embodiments, the extracellularrecording electrode pair as a third pair of electrodes. In still furtherembodiments at least one electrode of the pair of electro-stimulationelectrodes is also at least one electrode of the extracellular recordingelectrode pair thereby permitting electro-stimulation of excitable cellsand extracellular recording of attached cells using a same electrode atdifferent time points.

In regards to extracellular recording, the at least second pair ofelectrodes can be the extracellular recording electrode pair and theanalyzing module can be the extracellular recording amplifier.Preferably, the extracellular recording electrode pair includes arecording electrode and a reference electrode. In some embodiments therecording electrode has a diameter from about 10 μm to about 200 μm orfrom about 30 μm to about 100 μm. In some embodiments, the extracellularrecording electrode pair comprises a recording electrode and a referenceelectrode, further wherein a ratio of the area of reference electrode tothe area of recording electrode is selected from the group consisting of2 or more, 10 or more, 100 or more, 1,000 or more, and 10,000 or more.In further embodiments a second recording electrode is provided having asame diameter as a first.

In some extracellular recording embodiments, at least one electrode ofthe pair of electro-stimulation electrodes is also at least oneelectrode of a pair of impedance monitoring electrodes therebypermitting electro-stimulation of excitable cells and impedancemonitoring of attached cells using a same electrode at different timepoints and thus electro-stimulation together with simultaneous impedancemonitoring using as few as four total electrodes. In such embodiments,preferably the pair of impedance monitoring electrodes is a pair ofinterdigitated electrode structures, wherein each electrode structurecomprises a plurality of electrode elements. In variations of theextracellular recording embodiments, at least one electrode of the pairof impedance monitoring electrodes is the reference electrode.

In addition a device for monitoring excitable cells is also provided,the device including at least one well, each well having a bottom havinga nonconductive substrate, wherein the substrate has a surface suitablefor attachment of excitable cells; a pair of electro-stimulationelectrodes positioned on the substrate within the at least one well andconfigured to electro-stimulate the excitable cells; and at least asecond pair of electrodes positioned within the at least one well andselected from the group consisting of a pair of impedance monitoringelectrodes and extracellular recording electrode pair, wherein the pairof impedance monitoring electrodes is configured for monitoringcell-substrate impedance of cells attached to the substrate, and whereinthe extracellular recording electrode pair is configured for monitoringextracellular potential of cells attached to the substrate.

In regards to electro-stimulation electrodes a percentage of a surfacearea of the bottom of the at least one well occupied by the pair ofelectro-stimulation electrodes can vary but may be selected from thegroup consisting of 5% or more, 10% or more, 20% or more, 30% or more,50% or more, and 70% or more. In some embodiments each of theelectro-stimulation electrodes independently include an unbranchedelectrode structure or a branched electrode structure.

In some embodiments, the at least second pair of electrodes is the pairof impedance measurement electrodes. In such configurations eachelectrode within the pair of impedance measurement electrodes caninclude a plurality of electrode elements and can be configured as onehalf of a pair of interdigated electrodes. In some embodiments,electrode of the pair of impedance monitoring electrodes has a samesurface area.

In regards to extracellular recording configurations, the at leastsecond pair of electrodes can be the extracellular recording electrodepair. Preferably, the extracellular recording electrode pair includes arecording electrode and reference electrode; however, two or morerecording electrodes may be associated with one or more referenceelectrodes. Preferably each recording electrode has a diameter fromabout 10 μm to about 200 μm or from about 30 μm to about 100 μm. Theratio of the area of reference electrode to the area of recordingelectrode can vary but may be selected from the group consisting of 2 ormore, 10 or more, 100 or more, 1,000 or more, and 10,000 or more. Thereference electrode of the recording and reference electrode pair canhave a branched or an unbranched structure.

In some embodiments at least one electrode is shared between the pair ofelectro-stimulation electrodes and the at least second electrode pair.In such configurations, the at least second electrode pair is the pairof impedance measurement electrodes. In other configurations the atleast second electrode pair is the extracellular recording electrodepair.

In still further embodiments of the device the at least second electrodepair is the pair of impedance measurement electrodes, and device furtherincludes the extracellular recording pair as a third pair of electrodes.In such configurations, at least one electrode can be shared between thepair of impedance measurement electrodes and the recording and referenceelectrode pair.

In another aspect of the invention a method for monitoring excitablecells is provided, which includes providing a system forelectro-stimulating excitable cells and monitor impedance and/orextracellular potential of stimulated cells which has at least two pairsof electrodes; adding a sample of excitable cells to the device;electro-stimulating the excitable cells with electro-stimulationelectrodes; and monitoring electro-stimulated cells through the at leastsecond pair of electrodes. In some embodiments the excitable cells arecardiomyocytes or cardiomyocyte precursor cells. Electro-stimulation canbe performed at a plurality of time intervals, optionally at regulartime intervals.

When impedance monitoring, a pair of impedance monitoring electrodes cancommunicatively coupled to an impedance analyzer, thereby permitting thestep of monitoring electro-simulated cells to include monitoringimpedance of electro-stimulated cells. Preferably, impedance ismonitored in millisecond time resolution. The methods may also includeadding a compound suspected of affecting excitation contraction couplingof the excitable cells to the at least one well for analysis. Themethods may include calculating and comparing an impedance-basedparameter prior to and after adding the compound to identify whether achange occurs in the excitable cells in response to the compound. Insome embodiments, the impedance-based parameter is compared between atleast two different electro-stimulation intervals. Examples of suitableimpedance-based parameter include an impedance measurement, a cell indexcalculated from the impedance measurement, and a cell change indexcalculated from the cell index. Preferably, the impedance basedparameter is plotted as an impedance-based curve over time and the stepof comparing the impedance-based parameter between at least twodifferent electro-stimulation intervals includes comparingimpedance-based curves between at least to different electro-stimulationintervals. In some embodiments, the excitable cells are added to each ofat least two wells and the step of performing impedance measurements isperformed for each of the at least two wells, the method can thereforefurther include adding a compound suspected of affecting excitationcontraction coupling of the excitable cells to a first of the at leasttwo wells to form a test well and adding a control to a second of thelease two wells to form a control well, and comparing an impedance-basedparameter between the test well and control well. Again, suitableimpedance-based parameters can be the impedance measurement, a cellindex calculated from the impedance measurement, and a cell change indexcalculated from the cell index. Preferably, the impedance-basedparameter is plotted as an impedance-based curve over time and the stepof comparing the impedance-based parameter between the test well andcontrol well includes comparing the impedance-based curve of the testwell to the impedance based curve of the control well.

In some embodiments, the extracellular recording electrode pair iscommunicatively coupled to the an extracellular recording amplifier, andthe step of monitoring electro-simulated cells includes extracellularrecording of electro-stimulated cells. Preferably, the embodimentsinclude plotting extracellular potential from the extracellularrecording of electro-stimulated cells over time to form a fieldpotential curve. Extracellular recording can be performed before andafter the step of electro-stimulating the excitable cells.

In further embodiments, the method includes adding a compound suspectedof affecting excitation contraction coupling of the excitable cells tothe at least one well for extracellular recording. In such embodiments,extracellular potential of the excitable cells prior to and after addingthe compound can be compared to identify changes in extracellularpotential in response to the compound. Extracellular potential may beplotted over time to form a field potential curve for analysis.

In further embodiments, excitable cells are added to each of at leasttwo wells and the step of performing extracellular recordingmeasurements is performed for each of the at least two wells. In suchinstances, the method can include adding a compound suspected ofaffecting excitation contraction coupling of the excitable cells to afirst of the at least two wells to form a test well and adding a controlto a second of the lease two wells to form a control well, and comparingextracellular potential between the test well and control well.Extracellular recording can be performed before and after adding thecompound.

In some embodiments, a pair of impedance monitoring electrodes arecommunicatively coupled to the impedance analyzer and the extracellularrecording electrode pair is communicatively coupled to the extracellularrecording amplifier, and the step of monitoring electro-stimulated cellsincludes impedance monitoring and extracellular recording ofelectro-stimulated cells. In some embodiments at least one electrode isshared between two pairs of electrodes selected from the groupconsisting of the pair of electro-stimulation electrodes and the pair ofimpedance monitoring electrodes, the pair of electro-stimulationelectrodes and the extracellular recording electrode pair, and the pairof impedance monitoring electrodes and the extracellular recordingelectrode pair. In such configurations, the sharing of the electrode(s)is performed by switching communication to the at least one electrode.

In some embodiments the method include adding a compound suspected ofaffecting excitation contraction coupling of the excitable cells to theat least one well. Such methods may also include comparing animpedance-based parameter and extracellular field potential prior to andafter adding the compound to identify whether a change occurs in theexcitable cells in response to the compound. Preferably, theimpedance-based parameter is selected from the group consisting of animpedance measurement, a cell index calculated from the impedancemeasurement, and a cell change index calculated from the cell index. Instill further embodiments, the impedance based parameter is plotted asan impedance-based curve over time and the extracellular field potentialis plotted as field potential over time and the step of comparing theimpedance-based parameter and extracellular field potential between atleast two different electro-stimulation intervals includes comparingimpedance-based curves and comparing field potential curves between atleast two different electro-stimulation intervals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration showing a correlation of a cell indeximpedance parameter and field potential for a contraction-relaxationinterval in a primary neonatal rat cardiomyocyte.

FIGS. 2A-D depict different configurations of electrode arrays 10A-Dthat can perform electro-stimulation, impedance measurement andextracellular recording.

FIG. 3 depicts an exemplary system of a device 100, a combined analyzer200 having switchable electro-stimulation, impedance monitoring, andextracellular recording functions and computer 300.

FIGS. 4A-D depict examples of the graphical interface for software usedin data acquisition (FIGS. 4A-C) and data analysis (FIG. 4D).

FIG. 5 shows an exemplary cardio experimental work flow chart.

FIGS. 6A-B show results from extracellular recording of field potentialand impedance monitoring of HL-1 cells (FIG. 6A) and CorAT cells (FIG.6B).

FIGS. 7A-C show results from extracellular recording of field potentialand impedance monitoring of CorAT cells with the addition of Solatol at0 μM (FIG. 7A), 200 μM (FIG. 7B) or 400 μM (FIG. 7C).

FIGS. 8A-F show results from extracellular recording of field potentialand impedance monitoring of rat primary cardiomyocyte cells with theaddition of Quinidine at 0 μM (FIG. 8A), 2 μM (FIG. 8B), 4 μM (FIG. 8C),8 μM (FIG. 8D) or 16 μM (FIG. 8E). FIG. 8F shows an overlay of aninterval of ECR field potentials from each of FIGS. 8A-E. FIG. 8G showsan overlay of an interval of impedance from each of FIGS. 8A-E.

FIG. 9 shows changes in impedance and extracellular recording of fieldpotential of Cor.AT cells during electrostimulation and withoutelectrostimulation.

FIG. 10 shows changes in impedance and extracellular recording of fieldpotential of reat primary cardiomyocyte during electrostimulation andwithout electrostimulation.

FIGS. 11A-F shows a series of curves from an experiment where HL-1 cells(FIG. 11A), were electro-stimulated to induce measurable changes infield potential (FP) (FIG. 11B), which could then be modulated throughthe addition of different concentrations of the calcium channel blockerIsrapidine (FIGS. 11C-E). FIG. 11F being an overlay of field potentialcurves from FIGS. 11A-E.

FIGS. 12A-G show a series of curves from an experiment where HL-1 cells(FIG. 12A) where electrostimulated to induce measureable changes infield potential (FP) (FIG. 12B). A calcium channel activator was addedto culture and modulation of the field potential (FP) was detected (FIG.12C). The culture was paced use electro-stimulation (FIG. 12D). Additionof different concentrations of antagonist (FIGS. 12E-F) were added andmodulated the field potential (FP). FIG. 12G is an overlay of FIGS.12A-F)

FIGS. 13A-C show a series of curves from an experiment documenting theelectro-stimulation of CorAT cells and the pacing of cardiomyocyteswhere FIG. 13A overlays impedance (via cell index) and field potentialin response to electro-stimulation and after electro-stimulation stops.In FIG. 13B, impedance and field potential are split in that the upperpanel is impedance and the lower panel is field potential. FIG. 13C isan enlarged view showing a single interval and the relationship betweenimpedance and field potential.

FIGS. 14A-B show the electro-stimulation of rat primary cardiomyocytesand the impedance and field potential profiles in response to theregular pacing of cardiomyocytes.

FIGS. 15A-B show results from experiments monitoring the field potentialof neurons after treatment with bicuculline (FIG. 15A) or Glutamate(FIG. 15B).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As an introduction, we describe label-free methods forelectro-stimulating and monitoring excitable cells, such ascardiomyocytes, in vitro. It is an object of the invention to applyappropriate electro-stimulation signals for stimulating and/or pacingexcitable cells, together with extracellular recording of cells,impedance monitoring of cells, or parallel impedance monitoring andextracellular recording cells using microelectrodes to non-invasivelymonitor cells. To this end, the devices, systems and methods allow thecontinuous monitoring of cardiomyocyte viability overtime and canmonitor the interaction of compounds which ultimately result inpromoting loss of cardiomyocyte viability. Further, the devices systemsand methods permit high resolution monitoring of the beating cycle of apopulation of cardiomyocyte cells thus can detect changes in cellbeating, such as shifts in contraction or relaxation that would bedifficult to detect otherwise. As further introduction, an exemplaryplot of the relationship between impedance and extracellular recordingis shown in FIG. 1, where high resolution monitoring demonstrates thatcontraction and relaxation of cells, such as cardiomyocytes can beeffectively monitored using an impedance-based approach and morepreferably together with extracellular recording. In particular a peakin impedance (as measured in cell index) is followed by a peak in fieldpotential. According changes in either impedance or extracellularrecording provides insight as to changes occurring in the excitable cellpopulation. Still further, by incorporating short term impedancemonitoring with long term impedance monitoring the status excitable cellpopulation can be further studied, An implication of this highresolution monitoring discussed herein is that impedance monitoring andextracellular recording can be used as a high throughput approach toscreen for potential cardiotoxic affects of compounds. Further it alsoprovides approaches for monitoring embryonic stem cell development intocardiomyocytes and implications associated with genetic knock outs andtransgene expression at different developmental stages.

DEFINITIONS

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections thatfollow. Further, unless defined otherwise, all technical and scientificterms used herein have the same meaning as is commonly understood by oneof ordinary skill in the art to which this invention belongs. Allpatents, applications, published applications and other publicationsreferred to herein are incorporated by reference in their entirety. If adefinition set forth in this section is contrary to or otherwiseinconsistent with a definition set forth in the patents, applications,published applications and other publications that are hereinincorporated by reference, the definition set forth in this sectionprevails over the definition that is incorporated herein by reference.

As used herein, “biocompatible membrane or biocompatible surface” meansa membrane or surface that does not have deleterious effects on cells,including the viability, attachment, spreading, motility, growth, celldivision or cell beating.

As used herein, “biomolecular coating”, “biological molecule coating” or“coated with a biomolecule” refers a coating on a surface that comprisesa molecule that is a naturally occurring biological molecule orbiochemical, or a biochemical derived from or based on one or morenaturally occurring biomolecules or biochemicals. For example, abiological molecule coating can include an extracellular matrixcomponent (e.g., fibronectin, collagens), or a derivative thereof, orcan comprise a biochemical such as polylysine or polyornithine, whichare polymeric molecules based on the naturally occurring biochemicalslysine and ornithine. Polymeric molecules based on naturally occurringbiochemicals such as amino acids can use isomers or enantiomers of thenaturally-occurring biochemicals.

An “organic compound coating” or a “coating having an organic compound)as used herein refers to a coating on a substrate that includes anorganic compound. For example an organic compound may include a naturalligand or an agonist or an antagonist for a cell surface receptor.

An “electrode” is a structure having a high electrical conductivity,that is, an electrical conductivity much higher than the electricalconductivity of the surrounding materials, which in the presentinvention are typically nonconductive. An “extracellular recordingelectrode” or “recording electrode” or “ECR electrode” is such astructure used to detect electrical signal corresponding toextracellular field potential of the cell or cell population. Forinstance, a “recording electrode” may be used to monitor theextracellular field potential of a cardiomyocyte during the generationof membrane action potentials. A “reference electrode” is thecomplementary structure used to complete the electrical circuit duringextracellular recording. An “impedance electrode”, “impedance monitoringelectrode”, “impedance measurement electrode” or “impedance electrodestructure” is a structure, such as an electrode, used for impedancemonitoring. An “impedance electrode” may also operate as anextracellular recording electrode or electro-stimulation and thus mayprovide both impedance monitoring and extracellular recordingmeasurements or both impedance monitoring and electro-stimulation,albeit at different time points.

As used herein, an “electrode structure” refers to a single electrode,particularly one with a complex structure (as, for example, a spiralelectrode structure), or a collection of at least two electrode elementsthat are electrically connected together. All the electrode elementswithin an “electrode structure” are electrically connected.

As used herein, “electrode element” refers to a single structuralfeature of an electrode structure, such as, for example, a fingerlike orbranched projection of an interdigitated electrode structure. Anelectrode structure may have a plurality of electrode elements.

As used herein, a “unitary electrode structure” refers to a singleelectrode that is unbranched. That is, a “unitary electrode structure”does not include a plurality of electrode elements. For example, anunitary electrode structure may be of a circle, a square or othergeometry.

As used herein, a “pair of electrodes” or “electrode pair” is two ormore electrode structures that are constructed to have dimensions andspacing such that they can, when connected to a signal source, operateas a unit to generate an electrical field in the region of spaces aroundthe electrode structures. Preferred electrode structure units of thepresent invention can measure impedance changes due to cell attachmentto an electrode surface. Non-limiting examples of electrode structureunits are interdigitated electrode structure units and concentricelectrode structure units.

As used herein “electrode bus” is a portion of an electrode thatconnects individual electrode elements or substructures. An electrodebus provides a common conduction path from individual electrode elementsor individual electrode substructures to another electrical connection.In the devices of the present invention, an electrode bus can contacteach electrode element of an electrode structure and provide anelectrical connection path to electrical traces that lead to aconnection pad.

As used herein “electrode traces” or “electrically conductive traces” or“electrical traces”, are electrically conductive paths that extend fromelectrodes or electrode elements or electrode structures toward one endor boundary of a device or apparatus for connecting the electrodes orelectrode elements or electrode structures to a electro-stimulationpower source, an analyzer or amplifier, such as an impedance analyzer oramplifier, a voltage amplifier and the like. Electrical communication ofelectro-stimulation electrodes, impedance electrodes or extracellularrecording electrodes typically involves connection to a connection padusing an “electrode trace.”

As used herein “connection pad” is an area on an apparatus or a deviceof the present invention which is electrically connected to at least oneelectrode or all electrode elements within at least one electrodestructure on an apparatus or a device and which can be operativelyconnected to external electrical circuits (e.g., an impedancemeasurement circuit or a signal source or an extracellular voltagesignal amplifier). The electrical connection between a connection padand an impedance measurement circuit, an extracellular recording circuitor a signal source can be direct or indirect, through any appropriateelectrical conduction means such as leads or wires. Such electricalconduction means may also go through electrode or electrical conductionpaths located on other regions of the apparatus or device.

As used herein “Interdigitated” means having projections coming onedirection that interlace with projections coming from a differentdirection in the manner of the fingers of folded hands (with the caveatthat interdigitated electrode elements preferably do not contact oneanother).

As used herein, “at least two electrodes fabricated on the substrate”means that the at least two electrodes are fabricated or made orproduced on the substrate. The at least two electrodes can be on thesame side of the substrate or on the different side of the substrate.The substrate may have multiple layers, the at least two electrodes canbe either on the same or on the different layers of the substrate.

As used herein, “at least two electrodes fabricated to a same plane ofthe substrate” means that, if the nonconducting substrate has multiplelayers, the at least two electrodes are fabricated to the same layer ofthe substrate.

As used herein, “an electrode positioned on a different plane” refers tothe positioning of an electrode, typically an external electrode orreference electrode, above, below or along a different surface anglethan that which it is compared. An “electrode positioned on a differentplane” may be parallel to that of the first.

As used herein, “the . . . electrodes (or electrode structures) havesubstantially the same surface area” means that the surface areas of theelectrodes referred to are not substantially different from each other,so that the impedance change due to cell attachment or growth on any oneof the electrodes (or electrode structures) referred to will contributeto the overall detectable change in impedance to a same or similardegree as the impedance change due to cell attachment or growth on anyother of the electrodes (or electrode structures) referred to. In otherwords, where electrodes (or electrode structures) have substantially thesame surface area, any one of the electrodes can contribute to overallchange in impedance upon cell attachment or growth on the electrode. Inmost cases, the ratio of surface area between the largest electrode andthe smallest electrode that have “substantially the same surface area”is less than 10. Preferably, the ratio of surface area between thelargest electrode and the smallest electrode of an electrode array isless than 5, 4, 3, 2, 1.5, 1.2 or 1.1. More preferably, the at least twoelectrodes of an electrode structure have nearly identical or identicalsurface area.

As used herein, “the device has a surface suitable for cell attachmentor growth” means that the electrode and/or non-electrode area of theapparatus has appropriate physical, chemical or biological propertiessuch that cells of interest can viably attach on the surface and newcells can continue to attach, while the cell culture grows, on thesurface of the apparatus. However, it is not necessary that the device,or the surface thereof, contain substances necessary for cell viabilityor growth. These necessary substances, e.g., nutrients or growthfactors, can be supplied in a medium. Preferably, when a suspension ofviable cardiomyocytes, neuron cells, muscle cells or other excitablecells or other adherent cells such as epithelial cells or endothelialcells is added to the “surface suitable for cell attachment” when atleast 50% of the cells are adhering to the surface within twelve hours.More preferably, a surface that is suitable for cell attachment hassurface properties so that at least 70% of the cells are adhering to thesurface within twelve hours of plating (i.e., adding cells to thechamber or well that comprises the said device). Even more preferably,the surface properties of a surface that is suitable for cell attachmentresults in at least 90% of the cells adhering to the surface withintwelve hours of plating. Most preferably, the surface properties of asurface that is suitable for cell attachment results in at least 90% ofthe cells adhering to the surface within eight, six, four, two hours ofplating.

As used herein, “detectable change in impedance between or among saidelectrodes” (or “detectable change in impedance between or among theelectrode structures”) means that the impedance between or among theelectrodes (or electrode structures) would have a significant changethat can be detected by an impedance analyzer or impedance measurementcircuit when cells attach on the electrode surfaces. The impedancechange refers to the difference in impedance values when cells areattached to the electrode surface and when cells are not attached to theelectrode surface, or when the number, type, activity, adhesiveness, ormorphology of cells attached to the electrode-comprising surface of theapparatus changes. In most cases, the change in impedance is larger than0.1% to be detectable. Preferably, the detectable change in impedance islarger than 1%, 2%, 5%, or 8%. More preferably, the detectable change inimpedance is larger than 10%. Impedance between or among electrodes istypically a function of the frequency of the applied electric field formeasurement. “Detectable change in impedance between or among theelectrodes” does not require the impedance change at all frequenciesbeing detectable. “Detectable change in impedance between or among saidelectrodes” only requires a detectable change in impedance at any singlefrequency (or multiple frequencies). In addition, impedance has twocomponents, resistance and reactance (reactance can be divided into twocategories, capacitive reactance and inductive reactance). “Detectablechange in impedance between or among said electrodes” requires only thateither one of resistance and reactance has a detectable change at anysingle frequency or multiple frequencies. In the present application,impedance is the electrical or electronic impedance. The method for themeasurement of such impedance is achieved by, (1) applying a voltagebetween or among the electrodes at a given frequency (or multiplefrequencies, or having specific voltage waveform) and monitoring theelectrical current through said electrodes at the frequency (or multiplefrequencies, or having specific waveform), dividing the voltageamplitude value by the current amplitude value to derive the impedancevalue; (2) applying an electric current of a single frequency component(or multiple frequencies or having specific current wave form) throughsaid electrodes and monitoring the voltage resulted between or amongsaid electrodes at the frequency (or multiple frequencies, or havingspecific waveform), dividing the voltage amplitude value by the currentamplitude value to derive the impedance value; (3) other methods thatcan measure or determine electric impedance. Note that in thedescription above of “dividing the voltage amplitude value by thecurrent amplitude value to derive the impedance value”, the “division”is done for the values of current amplitude and voltage amplitude atsame frequencies. Measurement of such electric impedance is anelectronic or electrical process that does not involve the use of anyreagents.

As used herein, “arranged in a row-column configuration” means that, interms of electric connection, the position of an electrode, an electrodearray or a switching circuit is identified by both a row position numberand a column position number.

As used herein, “each well contains substantially same number . . . ofcells” means that the lowest number of cells in a well is at least 50%of the highest number of cells in a well. Preferably, the lowest numberof cells in a well is at least 60%, 70%, 80%, 90%, 95% or 99% of thehighest number of cells in a well. More preferably, each well containsan identical number of cells.

As used herein, “each well contains . . . same type of cells” meansthat, for the intended purpose, each well contains same type of cells;it is not necessary that each well contains exactly identical type ofcells. For example, if the intended purpose is that each well containsmammalian cells, it is permissible if each well contains same type ofmammalian cells, e.g., human cells, or different mammalian cells, e.g.,human cells as well as other non-human mammalian cells such as mice,goat or monkey cells, etc.

As used herein, “each well contains . . . serially differentconcentration of a test compound” means that each well contains a testcompound with a serially diluted concentrations, e.g., an one-tenthserially diluted concentrations of 1 M, 0.1 M, 0.01 M, etc.

As used herein, “dose-response curve” means the dependent relationshipof response of cells on the dose concentration of a test compound. Theresponse of cells can be measured by many different parameters. Forexample, a test compound is suspected to have cytotoxicity and causecell death. Then the response of cells can be measured by percentage ofnon-viable (or viable) cells after the cells are treated by the testcompound. Plotting this percentage of non-viable (or viable) cells as afunction of the dose concentration of the test compound constructs adose response curve. In the present application, the percentage ofnon-viable (or viable) cells can be expressed in terms of measuredimpedance values, or in terms of cell index derived from impedancemeasurement, or in terms of cell change indexes. For example, for a givecell type and under specific cellular physiological condition (e.g., aparticular cell culture medium), cell index can be shown to have alinear correlation or positive correlation with the number of viablecells in a well from which cell index was derived from the impedancemeasurement. Thus, in the present application, one can plot cell indexas a function of the dose concentration of the test compound toconstruct a “dose-response curve”. Note that, generally, cell index notonly correlate with the number of viable cells in the wells but alsorelate to the cell morphology and cell attachment. Thus plotting cellindex versus dose concentration provides information not only aboutnumber of cells but also about their physiological status (e.g. cellmorphology and cell adhesion). Furthermore, an important advantageoffered by the system and devices of the present invention is that in asingle experiment, one can obtain “dose-response curves” at multipletime points since the system allows for the continuous monitoring ofcells and provides impedance measurement at many time points over a timerange as short as a few minutes to as long as days or weeks.

As used herein, “sample” refers to anything which may contain a moietyto be isolated, manipulated, measured, quantified, detected or analyzedusing apparatuses, microplates or methods in the present application.The sample may be a biological sample, such as a biological fluid or abiological tissue. Examples of biological fluids include suspension ofcells in a medium such as cell culture medium, urine, blood, plasma,serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears,mucus, amniotic fluid or the like. Biological tissues are aggregates ofcells, usually of a particular kind together with their intercellularsubstance that form one of the structural materials of a human, animal,plant, bacterial, fungal or viral structure, including connective,epithelium, muscle and nerve tissues. Examples of biological tissuesalso include organs, tumors, lymph nodes, arteries and individualcell(s). The biological samples may further include cell suspensions,solutions containing biological molecules (e.g. proteins, enzymes,nucleic acids, carbohydrates, chemical molecules binding to biologicalmolecules).

As used herein, a “liquid (fluid) sample” refers to a sample thatnaturally exists as a liquid or fluid, e.g., a biological fluid. A“liquid sample” also refers to a sample that naturally exists in anon-liquid status, e.g., solid or gas, but is prepared as a liquid,fluid, solution or suspension containing the solid or gas samplematerial. For example, a liquid sample can encompass a liquid, fluid,solution or suspension containing a biological tissue.

A “compound” or “test compound” is any compound whose activity or director indirect effect or effects on cells is investigated in any assay. Atest compound can be any compound, including, but not limited to, asmall molecule, a large molecule, a molecular complex, an organicmolecule, an inorganic molecule, a biomolecule or biological moleculesuch as but not limited to a lipid, a steroid, a carbohydrate, a fattyacid, an amino acid, a peptide, a protein, a nucleic acid, or anycombination thereof. A test compound can be a synthetic compound, anaturally occurring compound, a derivative of a naturally-occurringcompound, etc. The structure of a test compound can be known or unknown.In one application of the present invention, a compound is capable of,or is suspected of, effecting cell adhesion or cell spreading. Inanother application of present invention, a compound is capable of, oris suspected of, stimulating or inhibiting cell adhesion or cellspreading. In still another application, a compound is capable of, or issuspected of, interacting with an ion channel. In still anotherapplication, a compound is capable of, or is suspected of, modulatingcardiomyocyte excitation contraction coupling or beating. In stillanother application, a compound is capable of, or is suspected of,interacting with cells (for example, binding to cell surface receptor,or inhibiting certain intracellular signal transduction pathway, oractivating cells).

A “known compound” is a compound for which at least one activity isknown. In the present invention, a known compound preferably is acompound for which one or more direct or indirect effects on cells isknown. Preferably, the structure of a known compound is known, but thisneed not be the case. Preferably, the mechanism of action of a knowncompound on cells is known, for example, the effect or effects of aknown compound on cells can be, as nonlimiting examples, effects on cellbeating, cell viability, cell adhesion, apoptosis, cell differentiation,cell proliferation, cell morphology, cell cycle, IgE-mediated cellactivation or stimulation, receptor-ligand binding, cell number, cellquality, cell cycling, cell adhesion, cell spreading, etc.

As used herein “Cell Index” or “CI” is a parameter that can derived frommeasured impedance values and that can be used to reflect the change inimpedance values. There are a number of methods to derive or calculateCell Index. The details of the method for calculating Cell Index,Normalized Cell Index, Delta Cell Index and cell change index can befound in U.S. patent application Ser. No. 10/705,447, filed on Nov. 10,2003; U.S. patent application Ser. No. 10/705,615, filed on Nov. 10,2003; U.S. patent application Ser. No. 10/987,73, filed on Nov. 12,2004; U.S. patent application Ser. No. 11/055,639, filed on Feb. 9,2005; U.S. patent application Ser. No. 11/198,831, filed on Aug. 4,2005; U.S. patent application Ser. No. 11/197,994, filed on Aug. 4,2005; U.S. patent application Ser. No. 11/235,938, filed on Sep. 27,2005, all of them are incorporated here by reference.

A “Normalized Cell Index” at a given time point is calculated bydividing the Cell Index at the time point by the Cell Index at areference time point. Thus, the Normalized Cell Index is 1 at thereference time point. Generally, for an assay involving treatment of thecells with compounds or with other bio-manipulation of the cells, thereference time point is the last time point for impedance measurementbefore the treatment of the cells.

A “delta cell index” at a given time point is calculated by subtractingthe cell index at a standard time point from the cell index at the giventime point. Thus, the delta cell index is the absolute change in thecell index from an initial time (the standard time point) to themeasurement time.

A “Cell Change Index” or “CCI” is a parameter derived from Cell Indexand “CCI” at a time point is equal to the 1^(st) order derive of theCell Index with respect to time, divided by the Cell Index at the timepoint. In other words, CCI is calculated as

${{CCI}(t)} = {\frac{{{CI}(t)}}{{{CI}(t)} \cdot {t}}.}$

As used herein “extracellular recording” refers to measuring, monitoringand/or recording of electric potential difference between two electrodestypically caused by ionic movement or ionic current through the media orsolution due to charge fluctuations across ion channels in a cell or ina group of cells. The cells are in the media or the solution. Incontrast to intracellular recording where the recording electrodes areplaced inside a cell through the cell membrane, the extracellularrecording electrodes are located outside of the cells.

Devices and Systems for Electro-Stimulation and Measurement of ExcitableCells

Provided herein are devices and systems that permit theelectro-stimulation as well as extracellular recording and/or impedancemonitoring of cells. In particular, an exemplary device includes atleast one well, each well having a bottom having a nonconductivesubstrate, the substrate having a surface suitable for attachment ofexcitable cells; a pair of electro-stimulation electrodes positioned onthe substrate within the at least one well and configured toelectro-stimulate the excitable cells; and at least a second pair ofelectrodes positioned within the at least one well. The at least secondpair of electrodes can be a pair of impedance monitoring electrodes oran extracellular recording electrode pair. In some embodiments, thesecond pair of electrodes is the pair of impedance monitoring electrodesand the extracellular recording pair is provided as a third electrodepair. The pair of impedance monitoring electrodes are configured tomeasure cell-substrate impedance of cells attached to the substrate, andthe extracellular recording electrode pair are configured to measureextracellular potential of cells attached to the substrate. Accordingly,the device permits the electro-stimulation of excitable cells, such ascardiomyocytes while various embodiments permit impedance monitoring,extracellular recording or both impedance monitoring and extracellularrecording of excitable cells.

The above is accomplished at least in part because while the substratehas a surface suitable for attachment of excitable cells, it has beenfound that the attachment of excitable cells on the substrate provides asuitable form for measuring extracellular potential through theextracellular recording electrode pair, and measuring cell-substrateimpedance through the pair of impedance monitoring electrodes, and stillfurther that the attached excitable cells can be electro-stimulated orpaced by electro-stimulation when appropriate electrical signals aredelivered to the pair of electro-stimulation electrodes. Thus, byproviding a device that permits alternating electro-stimulation coupledwith cell detection or measurement techniques the cell culture canmaintain a regular beating interval to test the cardiotoxic affects ofvarious compounds or monitor the development of cardiomyocyte precursorcells to cardiomyocytes.

Preferably, the device includes one or more fluid-impermeablereceptacles which serve as fluid containers or wells. Such receptaclesmay be reversibly or irreversibly attached to or formed within thesubstrate or portions thereof (such as, for example, wells formed as ina microtiter plate). Suitable fluid container materials compriseplastic, glass, or plastic coated materials such as a ceramic, glass,metal, etc. Descriptions and disclosure of devices that comprise fluidcontainers can be found in U.S. Pat. No. 7,470,533, herein incorporatedby reference for all disclosure of fluid containers and fluid containerstructures that can engage a substrate comprising electrodes forimpedance measurements, including their dimensions, design, composition,and methods of manufacture.

In some embodiments, commercial tissue culture plates can be adapted tofit the substrate. Bottomless plates may also be custom-made topreferred dimensions. The device may have any number of wells as desiredfor the particular experiment. For instance, the device may have 1 well,2 wells, 3 wells, 6 wells, 8 wells, 12 wells, 24 wells, 36 wells, 96wells, 384 wells, 1536 wells or the like. Preferably, well diameters arefrom about 1 millimeter to about 20 millimeters, more preferably fromabout 2 millimeters to about 8 millimeters at the bottom of the well(the end disposed on the substrate). The wells can have a uniformdiameter or can taper toward the bottom so that the diameter of thecontainer at the end in contact with the substrate is smaller than thediameter of the opposing end.

The surface of the substrate is suitable for cell attachment andoptionally growth. Preferably, the nonconducting substrate is planar,and is flat or approximately flat. The substrate may be constructed froma variety of nonconductive materials known in the present art,including, but not limited to, silicon dioxide on silicon,silicon-on-insulator (SOI) wafer, glass (e.g., quartz glass, lead glassor borosilicate glass), sapphire, ceramics, polymer, fiber glass,plastics, e.g., polyimide (e.g. Kapton, polyimide film supplied byDuPont), polystyrene, polycarbonate, polyvinyl chloride, polyester,polypropylene and urea resin. Preferably, the substrate is biocompatiblewith excitable cells such as cardiomyocytes; however, materials that arenot biocompatible can be made biocompatible by applying a biocompatibleor biomolecular coating with a suitable material, such as abiocompatible polymer or the like. Further, attachment or growth alongthe substrate or electrodes may be enhanced by pre-coating the substratewith a protein or compound that facilitates attachment or growth. Suchcompounds may be chosen according to techniques known in the cellularbiology arts; however, in some embodiments fibronectin is effective.Alternatively, the substrate may be chemically modified to displayreactive groups or binding moieties such as adhesion molecules thatenhance cell attachment, particularly ES cells that are cardiomyocyteprecursor cells or cardiomyocytes.

To improve efficiency of production, electrodes of the invention may beapplied to the substrate followed by joining the electrode appliedsubstrate to a plate of bottomless wells or may be applied to a wellalready having the substrate as a bottom. Electrodes may be formed fromlarger sheets of conductive metal, such as via laser ablation of ametallic film and may be applied directly to the substrate.Alternatively, electrodes may be printed on the substrate using printingtechniques such as those similar to ink-jet printing where a conductivefluid having ultraviolet (UV) curable monomers, polymers or compounds isprinted on the substrate, then a light source is applied to cure theapplied conductive fluid to form electrodes. The skilled artisan willappreciate that conductive material may be applied directly to a planarsubstrate or may be inserted into grooves laser ablated or formed intothe substrate surface. A glue, such as a UV curable glue, can be appliedbetween the substrate and electrode or above the electrode for addedsecurity. Further, when applying conductive fluids, it may be preferredto apply a mask prior to applying the fluid to further define theelectrode area.

Examples of suitable electrode configurations are shown in FIGS. 2A-2D,which depict examples where electrodes are shared between differentfunctional pairs, in particular, where two electrodes from the pair ofelectro-stimulation electrodes are shared with a pair of impedancemonitoring electrodes. Using this technical approach the pair ofimpedance monitoring electrodes can be used for electro-stimulating theexcitable cells when not measuring impedance. Thus, the electrode arraysprovide electrode structures for cell electro-stimulation,cell-impedance measurement and extra-cellular recording, as designed ona non-conductive substrate associated with a single well. Turning inmore detail to FIG. 2A an exemplary electrode array 10A includes anextracellular recording electrode pair formed from two round-circularextra-cellular recording electrodes 12A positioned at about the middleof the entire array 10A and a unitary one-piece reference electrode 14Apositioned at opposing ends of the array 10A. A pair of interdigitatedelectrodes (16A, 18A) formed from a plurality of electrode elements 19Afunction to electro-stimulate a cell population when switched to aelectro-stimulation mode and function to measure cell substrateimpedance when switched to an impedance monitoring mode. These sharedelectro-stimulation/impedance monitoring electrodes 16A, 18A are shownin a branched configuration. More specifically, a plurality of electrodeelements 19A of each electrode structure 16A, 18A are shown having acircle on line configuration.

As a nonlimiting example, particular specifications shown in FIG. 2A arethat the diameter of each of the two the recording electrodes 12A is 100μm; the distance between two recording electrodes 12A is 2.98 mm; thediameter of circles in the circle-on-line electrode elements 19A is 90μm, the center-to-center distance between two adjacent circle-on-lineelectrode elements 19A is 110 μm; and the gap between two sharedimpedance/electro-stimulation electrodes 16A, 18A and covering therecording electrodes 12A is ˜290 μm. Each of the electrodes 12A, 14A,16A, 18A are connected to a connection pad 26A via an electrical trace26A

A variety of other configurations have been developed includingelectrode structures for cell electro-stimulation, cell-impedancemeasurement and extracellular recording of cells, as designed on anon-conductive substrate associated with a single well. For example, inFIG. 2B, each electrode array 10B includes two round-circularextracellular recording electrodes 12B and a single unitary one-piecereference electrode 14B. Electrode structures 16B, 18B are used for bothelectro-stimulation and cell impedance monitoring albeit at differenttime points by electronic switching. In this exemplary embodiment, thediameter of each recording electrode 12B is 60 um; the distance betweentwo recording electrodes 12B is 2 mm; the diameter of circles in thecircle-on-line electrode elements 19B is 90 um, the center-to-centerdistance between two adjacent circle-on-line electrode elements 19B is110 um; the gap between two impedance-electrode-structures 16B, 18B andcovering the recording electrodes 12B is 290 um.

Still another configuration is shown in FIG. 2C, where each electrodearray 10C is configured on a non-conductive substrate associated with asingle well. The electrode array 10C includes a round-circularextra-cellular recording electrode 12C and a unitary one-piece referenceelectrode 14C. Electrode structures 16C, 18C perform both impedancemeasurement and electro-stimulation of cells at different time points byelectronic switching. In this example, a plurality ofcircle-on-circular-line electrode elements 19C form interdigitatedelectrode structures 16C, 18C. In this exemplary embodiment, thediameter of the recording electrodes 12C is 80 um; the distance betweentwo recording electrodes 12C is 1.44 mm; the diameter of circles in thecircle-on-line electrode elements 19C is 90 um, the center-to-centerdistance between two adjacent circle-on-line electrode elements 19C is110 um.

Still another configuration is shown in FIG. 2D, where an electrodearray 10D includes two round-circular extra-cellular recordingelectrodes 12D and a single unitary one-piece reference electrode 14D.Electrode structures 16D, 18D are used for cell impedance monitoring.Electrode Structures 20D, 22D are used for electro-stimulation and forcell impedance monitoring. In this exemplary embodiment, the diameter ofeach recording electrode 12D is 60 um; the distance between tworecording electrodes 12D is 2 mm; the diameter of circles in thecircle-on-line electrode elements 19D is 90 um, the center-to-centerdistance between two adjacent circle-on-line electrode elements 19D is110 um; the gap between two impedance-electrode-structures 16D, 22D andcovering the recording electrodes 12D is ˜290 um.

In regards to the device generally, configurations including two or morewells, preferably electrodes within each well of the device areindividually addressed, meaning that electrical traces and connectionpads of the arrays are configured such that an array can be connected toits power source, impedance amplifier or extracellular recordingamplifier independent of the operation of an array in a neighboring welland thus each well can operate using different electro-stimulationintervals, perform different measurements and the like without adverselyaffecting neighboring wells.

Electrical traces of conductive material used to connect each of theelectrodes to a corresponding connection pad can be fabricated of anyelectrically conductive material. The traces can be localized to thesurface of the substrate, and can be optionally covered with aninsulating layer. Alternatively the traces can be disposed in a secondplane of the substrate.

Turning to the pair of electro-stimulation electrodes generally, theelectrodes may be provided in a variety of shapes and configurations solong as the configuration permits electro-stimulation.Electro-stimulation can be accomplished using a variety of differentwaveforms such as rectangular, ramp, sinusoidal signals and the like.Either uni-polarity or bi-polarity signals can be used, with signalamplitude ranging from −2.5 V to +2.5 V. When pacing cardiomyocyteselectrical signal from between 1 V-2V was preferred while electricalsignal between about 1.1-1.3V was most preferred. In preferredembodiments, the electro-stimulation voltage can be controlled atvoltage resolutions up to 2 mV. Maximum frequency of the stimulationsignals could be up to 50 kHz.

Preferably, each of the pair of electro-stimulation electrodes of anelectrode array is connected to a separate connection pad, which ispreferably located at the edge of the substrate. Connecting the pair ofelectro-stimulation electrodes to the connection pads can be performedby applying electrical traces of conductive material therebetween. Thisfacilitates connection to a suitable power source by providing aninterface at which the power source can connect. Connection to theconnection pads is generally performed through the use of electricallyconductive pins, clips or the like.

One or more electro-stimulation electrodes can be shared with either theextracellular recording electrode pair or the pair of impedancemonitoring electrodes by timing the switching between the power sourcethat delivers the electro-stimulation signal through theelectro-stimulation electrodes and the extracellular recording amplifieror the impedance analyzer.

Turning now to the extracellular recording electrode pair generally,extracellular recording is conducted by amplifying and recordingelectrical voltage signals between a recording electrode(s) andreference electrode(s). Such electrical voltages are induced on theelectrodes as a result of ionic current or movement through cell culturemedia or solution supporting the cells during the experiment as a resultof opening and/or closing of different ion channels across cell membraneduring the action potential duration. In order to achieve improvedconsistency and reproducibility of the recorded voltage signals, it isdesirable to minimize the contribution of any electrical signal from thereference electrode to the recorded voltage signals and to ensure thatthe majority, if not all, of the recording voltage signals are derivedfrom that on the recording electrode. Thus, generally, it is desirableand it is recognized for the reference electrodes to have smallelectrode impedances. The small electrode impedance is achieved by usingreference electrodes with large effective surface areas by increasingthe ratio of the surface area of the reference electrodes to that ofrecording electrode by a factor of a hundred, even thousands of times.For example, FIG. 2A shows a schematic representation of such electrodepairs placed on a non-conductive substrate, including a small arearecording electrode 12A and a much larger area reference electrode 14A.As shown throughout FIGS. 2A-2D, preferably the reference electrode 14is positioned towards the perimeter of the electrode array 10; however,it must not physically contact the recording electrode 12.

The reference electrode generally, can be a unitary or unbranchedelectrode and may be of a simple geometry such as a circle, a square andthe like. In other embodiments, the reference electrode has a branchedconfiguration, which may result in a large surface for the referenceelectrode. In some embodiments, the ratio of the surface area of thereference electrode to that of the recording electrode is more than 2.In other embodiments, the ratio of the surface area of the referenceelectrode to that of the recording electrode is 10 or more than 10. Instill other embodiments, the ratio of the surface area of the referenceelectrode to that of the recording electrode is 100 or more than 100. Inother embodiments the ratio of the surface area of the referenceelectrode to that of the recording electrode is 1000 or more than 1000.In other the ratio of the surface area of the reference electrode tothat of the recording electrode is 10,000 or more than 10,000.

While it is preferable to simultaneously measure impedance and performextracellular recording, in some embodiments one or both electrodes ofthe pair of impedance measurement electrodes is shared with theextracellular recording electrode pair. When using impedance electrodesin the form of interdigated electrode structures having a plurality ofelectrode elements, typically the shared electrode would be used as areference electrode in the extracellular recording electrode pair. Thiscan be accomplished when the surface area of the impedance monitoringelectrode(s) is sufficiently larger than the surface area of therecording electrode to act as a reference electrode. The skilled artisanwill appreciate that by electrically switching a pair of interdigitatedimpedance electrodes from impedance monitoring to function as a singlereference electrode, the surface area ratio of the combinedinterdigitated electrodes to recording electrode would substantiallyincrease and thus may be preferable in some instances. Further, it isalso possible, though not preferred to utilize an impedance measurementelectrode as a recording electrode when the reference electrode issufficiently larger than the impedance measurement electrode. While notpreferred this approach is more likely when using impedance measurementelectrode configurations having a small working electrode and largecounter electrode as previously detailed in the art.

Turning now to the pair of impedance monitoring electrodes themselves,each pair includes two or more electrode structures that are constructedto have dimensions and spacing such that they can, when connected to animpedance analyzer, operate as a unit to generate an electrical field inthe region of spaces around the impedance electrode structures.Preferably the electric field is substantially uniform across the pairof impedance electrodes. In preferred embodiments, the pair of impedanceelectrodes includes two impedance measurement electrode structures, eachof which includes multiple electrode elements, or substructures, whichbranch from the electrode structure. In preferred embodiments, theelectrode structures in each pair have substantially the same surfacearea.

Each of the two complementary impedance monitoring electrode structuresof a pair connect to a separate connection pad that is preferablylocated at the edge of the substrate. In some embodiments, the arrayincludes two pairs of impedance monitoring electrodes separated by arecording electrode region where one or two recording electrodes arepositioned on a same plane of the substrate. In such embodiments it ispreferred that each electrode structure be assigned to a separateconnecting pad however the pairs could be electrically joined such as byconnection at a shared connection pad or through an electronic switch.

In some embodiments, for each of two or more pairs of impedancemonitoring electrodes in two or more wells, a first of the two impedancemonitoring electrode structures is connected to one of two or moreconnection pads, and the second of the two impedance monitoringelectrode structures is connected to another of the two or moreconnection pads.

Preferably, each pair of impedance monitoring electrodes of the devicehas an approximately uniform electrode resistance distribution acrossthe entire pair of electrodes. By “uniform resistance distributionacross the pair” is meant that when a measurement voltage is appliedacross the electrode structures of pair of impedance measurementelectrodes, the electrode resistance at any given location of the pairis approximately equal to the electrode resistance at any other locationon the pair. Preferably, the electrode resistance at a first location onthe pair and the electrode resistance at a second location on the pairdoes not differ by more than 30%. More preferably, the electroderesistance at a first location on the pair and the electrode resistanceat a second location on the same pair does not differ by more than 15%.Even more preferably, the electrode resistance at a first location onthe pair and a second location on the same pair does not differ by morethan 5%. More preferably yet, the electrode resistance at a firstlocation on the pair and a second location on the same pair does notdiffer by more than 2%.

Preferred arrangements for electrode elements that form the pair ofimpedance monitoring electrodes and gaps between the electrodes andelectrode buses in a given pair are used to allow all cells, no matterwhere they land and attach to the pair of impedance measurementelectrodes to contribute similarly to the total impedance changemeasured for the pair. Thus, it is desirable to have similar electricfield strengths at any two locations within any given pair of impedancemeasurement electrodes when a measurement voltage is applied to thepair. At any given location of the pair, the field strength is relatedto the potential difference between the nearest point on a firstelectrode structure of the pair and the nearest point on a secondelectrode structure of the pair. It is therefore desirable to havesimilar electric potential drops across the electrode elements andacross the electrode buses of a given pair. Based on this requirement,it is preferred to have an approximately uniform electrode resistancedistribution across the whole pair where the electrode resistance at alocation of interest is equal to the sum of the electrode resistancebetween the nearest point on a first electrode structure (that is thepoint on the first electrode structure nearest the location of interest)and a first connection pad connected to the first electrode structureand the electrode resistance between the nearest point on a secondelectrode structure (that is the point on the first electrode structurenearest the location of interest) and a second connection pad connectedto the second electrode structure.

Achieving an approximately uniform distribution across the pair ofimpedance measurement electrodes can be achieved, for example, by havingelectrode structures and electrode buses of particular spacing anddimensions (lengths, widths, thicknesses and geometrical shapes) suchthat the resistance at any single location on the pair is approximatelyequal to the resistance at any single other location on the pair. Inmost embodiments, the electrode elements (or electrode structures) of agiven pair will have even spacing and be of similar thicknesses andwidths, the electrode buses of a given pair will be of similarthicknesses and widths, and the electrode traces leading from a givenpair to a connection pad will be of closely similar thicknesses andwidths. Thus, in these preferred embodiments, a pair of electrodestructures is designed such that the lengths and geometrical shapes ofelectrode elements or structures, the lengths and geometrical shapes ofelectrode traces, and the lengths and geometrical shapes of buses allowfor approximately uniform electrode resistance distribution across thepair.

In some preferred embodiments of impedance measurement configurations,electrode structures comprise multiple electrode elements, and eachelectrode element connects directly to an electrode bus. Electrodeelements of a first electrode structure connect to a first electrodebus, and electrode elements of a second electrode structure connect to asecond electrode bus. In these embodiments, each of the two electrodebuses connects to a separate connection pad via an electrical trace.Although the resistances of the traces contribute to the resistance at alocation on the pair, for any two locations on the pair the traceconnections from the first bus to a first connection pad and from thesecond bus to a second connection pad are identical. Thus, in thesepreferred embodiments trace resistances do not need to be taken intoaccount in designing the geometry of the pair to provide for uniformresistances across the array.

When incorporating configurations having two or more wells, impedancemeasurement electrodes between electrode arrays of different wells canshare a connection pad. Preferably one of the impedance measurementelectrode structures of at least one of the electrode arrays of thedevice is connected to a connection pad that also connects to animpedance measurement electrode structure of at least one other of theelectrode arrays of another well device. Preferably for at least twoarrays of the device, each of the two or more arrays has a firstimpedance monitoring electrode structure connected to a connection padthat connects with an impedance measurement electrode structure of atleast one other electrode array of another well, and each of the two ormore arrays has a second impedance monitoring electrode structure thatconnects to a connection pad that does not connect with any otherelectrode structures or arrays of the device. Thus, in preferredconfigurations of a device there are at least two electrode arrays eachof which has a first impedance monitoring electrode structure that isconnected to a common connection pad and a second impedance monitoringelectrode structure that is connected to an independent connection pad.

Preferred arrays for devices of the present invention include at leastone pair of impedance measurement electrodes, each comprising twoelectrode structures, such as, in the form of a spiral configuration oran interdigitated configuration. Preferably the pair of impedancemonitoring electrodes are fabricated on the substrate, in which the paircomprises two impedance monitoring electrode structures, each of whichcomprises multiple circle-on-line electrode elements, in which theelectrode elements of one electrode structure alternate with theelectrode elements of the opposite electrode structure. The pair ofimpedance monitoring electrodes may be provided in configurations, suchas interdigitated, circle-on-line, diamond-on-line, concentric,sinusoidal and castellated.

A central advantage to systems described herein is that they canincorporate two separate and complete recording systems into one singleinstrument, namely, an extracellular signal recording system andimpedance measurement system. This is accomplished by collecting fieldpotential, which is a local readout such as by an extracellularrecording electrode and impedance monitoring, which is an entire wellreadout by at least one pair of impedance electrodes respectively. Inaddition, with the capability of simultaneous recording of extracellularrecording and impedance, the interaction of the results obtained fromthese separate recording system can be used to support each other.

Parallel impedance-based monitoring as well as extra-cellular recordingbased monitoring of cardiomyocytes, under the condition ofelectro-stimulation of cardiomyocytes, fills a major technological gapin monitoring cardiomyocytes in vitro. At present to our knowledge thereare only a few technologies that can monitor cardiomyocyte populationfunction in vitro, especially with regards to cardiotoxicity and arelimited in their throughput. In addition to functional monitoring ofcardiomyocyte beating the current invention offers several other majorbenefits which are worth discussing. Among these include the impedancesystem described here can monitor cardiomyocytes for short durations,milliseconds to minutes and long durations, several hours to days.Therefore, both short term and long term effects of drugs not only oncardiomyocyte beating, but viability and changes in morphology andadhesion can also be assessed. This feature is especially importantbecause certain compounds such as β-2 adrenergic receptor agonists, wellknown and characterized modulators of heart function in vivo and invitro, can induce long term hypertrophic responses in cardiomyocytes,which is associated with elongated morphology of the cells. Also, thesystems of the present invention can also record cardiomyocyte actionpotentials or field potentials, allowing detailed electrophysiologicalanalysis and assessment of cardiomyocytes for relatively short durationat a time. Furthermore, the ability of electro-stimulatingcardiomyocytes allow for electrophysiological measurement and impedancemeasurement of otherwise non-beating cardiomyocytes. The synchronizationof electro-stimulation with extra-cellular potential recording and cellimpedance measurement would allow better and precise control ofinitiation timing points for excitation process and allow morereproducible measurement of extra-cellular potential and cellimpedances.

In view of the above with or without applying electro-stimulationsignals to stimulate or pace cardiomyocytes, impedance readout can beused to monitor the morphological or differentiative behavior ofcardiomyocytes in vitro. Certain treatments can induce changes inmorphological behavior of cardiomyocytes, such as inducing hypertrophywhich is associated with cardiomyocyte elongation and expansion. Becauseimpedance monitoring can detect changes in cell morphology, it can beused to for detection of hypertrophy in cardiomyocytes.

In cardiac culture preparation, impedance monitoring reflects the cellgrowth, which can be correlated through an impedance-based curve,namely, a cell index curve and contraction/relaxation function byimpedance beating signals; while extracellular recording reflectscardiac excitation and conduction by field potential signals. Further,this the invention provides the ability to monitor viability ofcardiomyocytes, the field potential of cardiomyocytes, or theexcitation-contraction coupling or beating and morphological anddifferentiative aspects of cardiomyocytes in a label-free manner andreal-time manner.

While the invention is primarily discussed in regards to cardiotoxicityand monitoring cardiomyocytes, the devices and systems can also be usedto study a neuronal culture preparation, in which impedance monitoringreflects the cell growth, which can be correlated through a cell indexgrowth curve. In addition as with electro-stimulation of cardiomyocytes,an impedance electrode can also serve for stimulation in the neuronalcell preparation, while evoking a response for network stimulation byeither chemical or electrical could be recorded at the extracellularrecording electrode(s). Theoretically, the configurations could also beused for any excitable cell cultures and tissue slices, so the systempossesses high capacity for multiple applications.

An exemplary system for electro-stimulation, impedance monitoring andextracellular recording of excitable cells requires electricalconnection of a device to a suitable a suitable electro-stimulationpower source, impedance analyzer, and/or extracellular recordingamplifier. With the above in mind, the invention provides a system formonitoring excitable cells, which includes a device having at least onewell, each well having a bottom having a nonconductive substrate,wherein the substrate has a surface suitable for attachment of excitablecells; a power source configured to deliver an electrical signal capableof electro-stimulating excitable cells; and at least one analyzingmodule for measuring an electrical property from electro-stimulatedexcitable cells, characterized in that each well includes a pair ofelectro-stimulation electrodes configured to receive the electricalsignal from the power source thereby delivering an electro-stimulatingsignal to the well for electro-stimulation of excitable cells attachedto the substrate; and at least a second pair of electrodescommunicatively coupled to the at least one analyzing module, which isselected from the group consisting of a pair of impedance monitoringelectrodes communicatively coupled to the at least one analyzing modulein the form of an impedance analyzer thereby permitting impedancemonitoring of excitable cells attached to the substrate, and anextracellular recording electrode pair communicatively coupled to the atleast one analyzing module in the form of an extracellular recordingamplifier thereby permitting extracellular recording of excitable cellsattached to the substrate.

Referring to FIG. 3, in some embodiments a the power source 220,impedance analyzer 230 and extracellular recording amplifier 240 arecombined into a combined analyzing module 200, which is operablyconnected to a multiwall device 100 for electro-stimulation andmonitoring of excitable cells. An electronic switch 210 can selectivelyswitch electrical connection with at least one electrode of the device100 between the power source 220 and impedance analyzer 230, between thepower source 220 and extracellular recording amplifier 240, and/or theimpedance analyzer 230 and extracellular recording amplifier 240,thereby providing numerous combinations. The combined analyzing module200 can then be coupled to a computer 300 loaded with software toexecute programming or analyze data.

Accordingly, in some embodiments of the invention, a system formonitoring excitable cells is provided, which includes a device forelectro-stimulation and monitoring of excitable cells, a power sourcecapable of delivering an electrical signal to a pair ofelectro-stimulation electrodes that stimulates excitable cells; at leastone analyzing module in the form of an impedance analyzer capable ofmonitoring cell substrate impedance of cells through the pair ofimpedance monitoring electrodes or an extracellular recording amplifiercapable of recording extracellular potentials of cells through theextracellular recording electrode pair. The system is configured toelectro-stimulate the excitable cells at a different time compared tomeasurement from the at least second pair of electrodes.

Electro-stimulation is accomplished by an electrical signal deliveredfrom the power source to the electro-stimulation electrodes and toattached cells, which can be in the form of a series of electricalpulses. That is, while impedance monitoring and extracellular recordingmay incorporate different spectra, waveforms or the like that cancontinue over an extended time, electro-stimulation is typicallyperformed more quickly in a fast on-off approach followed by a delay inthe off state to permit measurement of the excitable cells withoutinterference of the electro-stimulation signal. The skilled artisan willappreciate that in some instances the electro-stimulation signal may beat least partially filtered to prevent interference from a lowerintensity state. Preferably, electro-stimulation of the excitable cellsis performed at a plurality of time intervals. Most preferably, the timeintervals are at regular time intervals. As a nonlimiting example,electro-stimulation can be performed by delivering a biphasic pulse ofabout 0.8-1.5 ms, more preferably about 1 ms and at an output voltage of1-2 V or more preferably about 1.2 V.

An extracellular recording amplifier is communicatively coupled to theextracellular recording electrode pair for electrical communication topermit amplifying and recording electrical voltage signals between therecording electrodes and reference electrodes. That is, extracellularrecorded voltage signals are recorded as the difference in theelectrical potentials between the recording electrode and referenceelectrode. Such electrical voltages are induced on the electrodes as aresult of ionic current or movement through cell culture media orsolution supporting the cells during the experiment as a result ofopening and/or closing of different ion channels across cell membraneduring the action potential duration. In order to achieve improvedconsistency and reproducibility of the recorded voltage signals, it isdesirable to minimize the contribution of any electrical signal from thereference electrode to the recorded voltage signals and to ensure thatthe majority, if not all, of the recording voltage signals are derivedfrom that on the recording electrode.

Generally, it is desirable and it is recognized for the referenceelectrodes to have small electrode impedances. The small electrodeimpedance is achieved by using reference electrodes with large effectivesurface areas by increasing the ratio of the surface area of thereference electrodes to that of recording electrode by a factor of ahundred, even thousands of times. Such small electrode geometry hasadvantages of recording the electrical potential generated by a smallnumber of the cells located on the recording electrodes. Actionpotentials from such a small number of the cells tend to be synchronizedor nearly synchronized, allowing for a better time resolution forrecording extracellular potential and for resolving different featuresof the recorded potential. However, one limitation of such extracellularrecording is that due to small area of such electrodes, there tends tobe large variations in recorded signals between different recordedelectrodes of the same geometry in the same wells (if multiple recordingelectrodes are positioned inside a single well) or different wells. Inparticular, if insufficient number of the cells is added to the well tocover all the recording electrodes, it is possible that some recordingelectrodes may not show any recorded signal or only show recordedsignals of very small magnitude. Furthermore, depending on exactdistribution or locations of the cells on the recording electrodes,different recording electrodes may show significantly differentextracellular potential waveforms. For this reason, it may be preferableto have at least two recording electrodes positioned at about the middleof the array an in some instances more. Extra-cellular potentials fromeach such recording electrode can amplified and recorded separately. Theuser can then pick and choose appropriate signal waveforms recorded fromindividual recording electrodes for data analysis. Alternatively or incombination, a preferred approach is to culture a layer of excitablecells such that the layer covers all recording electrodes.

In the system for monitoring impedance of excitable cells the impedanceanalyzer is communicatively coupled to impedance monitoring electrodesto monitor impedance. In some embodiments, the impedance analyzer iscapable of measuring impedance between 0.1 ohm and 10⁵ ohm in frequencyrange of 1 Hz to 1 MHz. The impedance analyzer is preferably capable ofmeasuring both resistance and reactance (capacitive reactance andinductive reactance) components of the impedance. In a preferredembodiment of the above system, the impedance analyzer is capable ofmeasuring impedance between 1 ohm and 10³ ohm in frequency range of 1.00Hz to 300 kHz.

In preferred embodiments the impedance analyzer is capable of impedancemeasurements at millisecond time resolution. The required or desiredtime resolution may vary depending on the excitation cycle of theexcitable cell. Excitable cells having shorter excitation cycles orpacing electro-stimulation more quickly, would tend to require fastertime resolution. In some embodiments 500 millisecond time resolution issufficient, such that at least two consecutive impedance measurementsare between about 300 milliseconds and about 500 milliseconds apart. Inpreferred embodiments, impedance measurement at millisecond timeresolution includes at least two consecutive impedance measurements lessthan 100 milliseconds apart. In some instances the at least twoconsecutive impedance measurements are less than 50 milliseconds or lessthan 40 milliseconds apart. In some instances the at least twoconsecutive impedance measurements are less than 20 milliseconds apart.In some instances at least two consecutive impedance measurements areless than 10 milliseconds apart. In some instances millisecond timeresolution includes two consecutive impedance measurements between 1millisecond and 5 milliseconds, between 5 milliseconds and 10milliseconds, between 10 milliseconds and 20 milliseconds, between 20milliseconds and 40 milliseconds, or between 40 milliseconds and 50milliseconds apart. In some instances millisecond time resolutionincludes at least two consecutive impedance measurements between 50milliseconds and 100 milliseconds apart. In some instances millisecondtime resolution includes at least two consecutive impedance measurementsbetween 100 milliseconds and 150 milliseconds or between 150 and 300milliseconds apart.

Achieving millisecond time resolution can be achieved by using fastprocessing electronic chips for analogue-to-digital conversion, forparallel digital signal processing and data calculation withfield-programmable gate array (FPGA) and for fast communication betweenthe impedance measurement circuitry and software. Another example ofincludes the use of multiple analogue-to-digital (AD) conversionchannels so that analog electronic signals from multiple channels can beconverted to digital signals simultaneously. Such parallel AD conversionis important, particular for the system having multiple wells, each ofwhich's measurement time resolution is required to be in the millisecondresolution.

In addition, when using a device having multiple wells, the software canissue a command for measuring multiple wells' impedances. Themeasurement circuitry would simultaneously or nearly simultaneouslyperform signal conversion, signal processing and impedance calculationfor multiple wells. The multiple impedance data for the multiple wellswould be sent over the communication lines to the computer sequentiallywith one well's data at the same time or simultaneously with more thanone well's data being sent at a time. In this “measurement ofmultiple-wells' impedances at a time” mode, the system may be performingmultiple tasks simultaneously, for example, while one well's impedancedata is being measured and calculated, another well's impedance data maybe communicated and sent over the communication lines to the computer.

With millisecond time resolution for impedance measurement, it becomespossible to resolve individual beating cycles of cardiomyocytes culturedon electrodes and study the excitation, contraction and release ofbeating cells. Whilst theoretically one needs at least two data pointsfor each beating cycle, in practice more than 2 data points are neededfor each beating cycle. For example, if cells have a beating rate of 60beats per minute, i.e, one beat per second. It would be preferred tohave a time resolution of at least 200 milliseconds so that each beatingcycle consists of 5 data points. More preferably, the measurement timeresolution is 100 milliseconds. Still more preferably, the timeresolution is 50 milliseconds or less.

Cell-substrate impedance monitoring at millisecond time resolution canbe used to efficiently and simultaneously perform multiple assays byusing circuitry of the device station to digitally switch from recordingfrom measuring impedance over an array in one well to measuringimpedance over an array in another well. Similarly, groups of wells maybe monitored simultaneously and switching between occur betweendesignated groups. In one embodiment of the above system, the systemunder software control is capable of completing an impedance measurementfor an individual well at a single frequency within milliseconds, suchas less than 100 milliseconds, less than 40 milliseconds, less than 20milliseconds, less than 10 milliseconds or between 1 millisecond and 40milliseconds. In some embodiments the user may choose the frequency ofmeasurement for millisecond time resolution.

As discussed above, one or more electrodes in a pair of electrodes canbe shared with another pair of electrodes. Sharing of electrodes canaccomplished at least in part by switching the communicative couplingfor electrical connection between the power source forelectro-stimulation and either the impedance analyzer or extracellularrecording amplifier. Alternatively, sharing of electrodes can beaccomplished at least in part by switching the communicative couplingfor electronic connection between the impedance analyzer and theextracellular recording amplifier. In each instance, switching ispreferably performed through an electronic programmable switch and isprovided together with the power source, impedance analyzer andextracellular recording amplifier in a combined analyzer, which itselfcan be coupled to a computer for performing measurement instructions ordata analysis.

In some embodiments a combined analyzer or electromechanical apparatusor assembly capable of interfacing multiwell devices can include one ormore platforms or one or more slots for positioning one or moremultiwell devices, such as in the form of a device station withcarriage. The one or more platforms or one or more slots can comprisesockets, pins or other devices for electrically connecting the device tothe device station such as interaction through connection pads. Thesystem can be configured such that multiwell devices can be positionedin a tissue culture incubator during electro-stimulation, extracellularrecording or impedance monitoring while the computer and optionallycombined analyzer are located outside the tissue culture incubator. Thiscan be accomplished through the electrical connection of the devicethrough a device station that itself is inside the incubator andelectrically coupled to a combined analyzer that is outside of theincubator. Alternatively, the combined analyzer can be within theincubator and communicate with the computer that is outside of theincubator.

The device station or electromechanical apparatus or assembly capable ofinterfacing multiwell devices includes electronic circuitry that connectto the power source, impedance analyzer, and/or extracellular recordingamplifier and can incorporate electronic switches that can switch on andoff connections to each of the two or more pairs of electrodes within awell of the multiwell devices used in the system. The switches of thedevice station or electromechanical apparatus or assembly capable ofinterfacing multiwell devices are controlled by a software program, eachof which preferably provides millisecond time resolution.

The systems of the invention are typically controlled through commandssent from a computer operably connected to the power source, impedanceanalyzer and extracellular recording amplifier. The computer istypically loaded with software that provides two functions. A first isdata acquisition and a second is data analysis. The software usesgraphical interfaces for ease of use.

The data acquisition software is used to define the experiment, setuprecording steps, monitor the raw data, adjust settings and to collectraw data files. An example of graphical interface for data acquisitionis shown in FIGS. 4A-C, where FIG. 4A shows an interface for enteringexperimental notes such as experiment name, purpose or the like; FIG. 4Bshows a well layout where HL-1 atria cells are seeded at differentdensity from 10,000 in column 1 to 40,000 in column 4, and FIG. 4C showswells specific settings for, electro-stimulation, impedance monitoringand extracellular recording.

Preferably, the software can also analyze impedance data andextracellular recording data. An exemplary graphical interface for dataanalysis is provided in FIG. 4D, which shows a file structure “tree” atthe left side for file management, single or multiple channels of rawdata file displayed in the middle section of the window and the analysisresult displayed at the right side of the window. In addition, the dataanalysis software also has the ability to do single field potentialwaveform analysis.

An overview of an exemplary experiment and how it interacts with thesoftware can be seen in FIG. 5, where a first step involves cellpreparation 400, such as culturing cells, proceeds through raw datacollection 500 using electro-stimulation 510 if extracellular recordingor impedance monitoring does not result in desired activity, such aspoor beating of cardiomyocytes. After raw data collection 500 dataanalysis 600 is conducted such as correlation of Impedance and ECR data620. Finally reports 700 are generated providing the results from theimpedance and extracellular recording results in desired formats.

In preferred embodiments, the software can calculate an impedance-basedparameter, such as cell index (CI) for one or more time points for oneor more wells of the multiwell device through suitable programming. Insome preferred embodiments, the software can also calculate a cellchange index (CCI) from impedance measurements of one or more wells ofthe multiwell device. The software can preferably generate plots ofimpedance-based parameters over time, such as but not limited toimpedance-based curves selected from impedance measurements, CI, or CCI.The software may perform other analysis as well, such as calculate cellnumber from CI, generate dose-response curves based on impedance data,calculate IC values based on impedance values, and calculate kineticparameters of the excitation cycle cell based on impedance-basedparameters and impedance-based curves. In some embodiments the beatingcycle of a cardiomyocyte population is determined, which may includeinitiation and decay of individual beats. Peaks may be derived from thedetection of vectors associated with initiation of beating or beatingdecay. Peaks may be derived with other methods. In further embodimentsthe change in beating cycle of a cardiomyocyte population is determinedin response to a stimulus such as administration of a compound to thecells. The software of the impedance monitoring system can also storeand display analyses of the data, such as calculated impedance valuesimpedance-based parameters and kinetic parameters derived therefrom,Data can be displayed on a screen, as printed data, or both. Data may bestored on a hard drive for exportation into compatible programs forfurther analysis or data storage

Methods of Monitoring Cardiomyocyte Beating, Viability, Morphology andElectrophysiological Properties

The invention provides methods of monitoring excitable cells, methodsfor monitoring cardiomyocyte beating, viability, morphology, assessingcardiotoxic affects associated with administering of one or morecompounds to a population of cardiomyocyte or cardiomyocyte precursorcells. The methods include obtaining a sample of excitable cells,electro-stimulating the excitable cells; and impedance monitoring and/orextracellular recording of the excitable cells. Preferably, impedancemonitoring and extracellular recording is performed simultaneously toachieve simultaneous real time measurements.

It is well established that certain pharmacological treatments anddisease conditions can result in cardiac hypertrophy or atrophyculminating in changes in the morphology of cardiomyocyte. The methodsand systems herein have developed a system for monitoring cell substrateimpedance to precisely measure and quantify these changes in cellmorphology and shape.

In addition, the present invention also discloses an approach formonitoring extracellular field potential, which is an electricalpotential produce by cells that is outside of the cell. However, asshown in the examples, extracellular recording also provides insight asthe activity of ion channels, which are of central interest whenassessing cardiotoxicity of potential therapeutics.

In view of the above, a method of monitoring excitable cells is provide,which includes providing a system for electro-stimulation, impedancemonitoring and extracellular recording of excitable cells; adding asample of excitable cells to the device; electro-stimulating theexcitable cells; and impedance monitoring and extracellular recording ofthe excitable cells.

In a related embodiment, a method of monitoring excitable cells isprovided, which includes providing a system for electro-stimulation andimpedance monitoring of excitable cells; adding a sample of excitablecells to the device; electro-stimulating the excitable cells; andperforming impedance measurements of the excitable cells.

In another related embodiment, a method of monitoring excitable cells isprovided, which includes providing a system for electro-stimulation andextracellular recording of excitable cells; adding a sample of excitablecells to the device; electro-stimulating the excitable cells; andperforming extracellular recording of the excitable cells.

Among the cells that may be monitored include any excitable cells, suchas primary cardiomyocytes harvested from animal tissue, culturedcardiomyocytes, cardiomyocyte precursor cells, embryonic stem cells,embryonic stem cell derived cardiomyocytes, cardiomyocyte cell lines,enriched cardiomyocyte cells and the like. Alternatively the inventionhas also shown that neuronal cells may also be used.

Obtaining or harvesting suitable excitable cells may be performed usinga variety of harvesting, collecting, culturing, purifying, enriching ordifferentiating techniques known in the art to which the inventionbelongs. While the device can detect changes, such as impedance changeand extracellular potential in a single cell, preferably samples containa plurality of cells so that the cells will join together forcommunication with one another, such as through gap junctions. Clearchanges in electrocellular recording and impedance measurement have beenidentified using population of about 10,000-14,000 cells; however, thisis not intended to be limiting.

Cells may be placed into culture then immediately stimulated ormeasured; however, it has generally been preferable to culture cellssuch that they attach to the substrate prior to stimulation. Morepreferably, cells are grown or differentiated on the substrate for asufficient time in that they join to form a cell layer over therecording electrodes of the extracellular recording electrode pair andover the pair of impedance monitoring electrodes. In some embodiments,electro-stimulation is performed within 1 hour of placing cells in thedevice; however, the substrate forming the bottom of the well has beenshown suitable for long term culturing and thus cells may be cultured asdesired, such as for more than 5 days, more than 10 days, more than 15days, more than 20 days or even more than one month depending on theinterests of the experiment and regular cell culture maintenance.

Further, impedance monitoring may be used to assess how well cells areattached to the substrate, the viability of the cells or to assess thequantity of cells in the sample prior to electro-stimulation, to assessgrowth curves or the like. Further, impedance or extracellular recordingmay be used to determine whether electro-stimulation is necessary ordesired. In some instances cardiomyocytes derived from embryonic stemcells will begin to beat spontaneously, which can be detected ormeasured through impedance and extracellular recording. However, evencells that being spontaneous beating can be electro-stimulated toestablish a more regular beating or a beating at an increased frequency.

Excitable cells are electro-stimulated through applying a signal via thepair of electro-stimulation electrodes. As indicated above, the pair ofelectro-stimulation electrodes may be a dedicated pair of electrodes,preferably one or more electrodes within the pair of electro-stimulationelectrodes may be shared between the pair of impedance measurementelectrodes or the extracellular recording electrode pair. Sharing of theelectro-stimulation function is permitted in part because of the fastswitching between connectivity to the power source forelectro-stimulation and the impedance analyzer or between the powersource for electro-stimulation and the extracellular recordingamplifier. While the particular specifications of a device can vary,stimulation is generally accomplished by applying voltage in a range ofabout 1-2 V for about 1 ms, at an interval of every 1-1.5 seconds or so.Particularly encouraging results were obtained when applying 1.1 V for 1ms every 1.2-1.3 s.

When providing two or more wells, each well may be pulsed together suchthat electro-stimulation occurs simultaneously in more than one well orpulses may be staggered such that two or more wells are pulsed withdifferent at different interval. Electro-stimulation intervals can beselected through a software and computer interface.

When performing impedance monitoring, preferably, impedance is measuredat millisecond time resolution. For example, resolution on the order of1-10 ms provides high resolution of beating of cardiomyocytes. Impedancemeasurements may themselves be compared; however, in preferredembodiments a cell index is calculated from impedance measurements as animpedance parameter and plotted over time as an impedance-based curve orcell index curve. Accordingly, a cell index curve may be used as animpedance-based curve for comparison, such as by comparing cell indexcurves over time. In other embodiments, cell change index is calculatedfrom cell index and plotted as an impedance based curve. Accordingly,cell change index may be used as impedance-based parameter forcomparison, such as by comparing cell change index curves over time. Theskilled artisan will appreciate that when comparing impedance-basedcurves, a same type of curve will be compared. For example, comparisonwould involve comparing cell index curves to one another, cell changeindex curves to one another and the like.

While impedance measurements themselves can be compared, theircomparison is complicated by culture conditions, such as differencesbetween cell populations. Accordingly a number of calculations have beenestablished previously that result in values that permit improvedcomparison. Among these are cell index (CI) and cell change index (CCI),each of which has been discussed in detail in the following US patents:U.S. Pat. No. 7,470,533, U.S. Pat. No. 7,459,303, U.S. Pat. No.7,192,752, U.S. Pat. No. 8,026,080, U.S. Pat. No. 7,560,269, U.S. Pat.No. 8,263,375, U.S. Pat. No. 7,468,255, U.S. Pat. No. 8,206,903 and U.S.Pat. No. 8,420,363. Accordingly, the use of cell index and cell changeindex in the formation of an impedance-based curve is well establishedin the art. Further, these documents can be consulted for method ofcalculating cell index (CI) or cell change index (CCI). However, a briefoverview introduction is provided below.

The cell index (CI) obtained for a given well reflects how many cellsare attached to the electrode surfaces in this well and how well cellsare attached to the electrode surfaces in the well. In this case, a zeroor near-zero “cell index” indicates that no cells or very small numberof cells are present on or attached to the electrode surfaces. In otherwords, if no cells are present on the electrodes, or if the cells arenot well-attached onto the electrodes cell index=0. A higher value of“cell index” indicates that, for same type of the cells and cells undersimilar physiological conditions, more cells are attached to theelectrode surfaces. Thus Cell Index is a quantitative measure of cellnumber present in a well. A higher value of “cell index” may alsoindicate that, for same type of the cells and same number of the cells,cells are attached better (for example, cells spread out more, or celladhesion to the electrode surface is stronger) on the electrodesurfaces.

In other embodiments, a normalize cell index is calculated from the cellindex and plotted as an impedance based curve. A “normalized cell index”at a given time point is calculated by dividing the Cell Index at thetime point by the Cell Index at a reference time point. Thus, theNormalized Cell Index is 1 at the reference time point. Normalized cellindex is cell index normalized against cell index at a particular timepoint. In most cases in the present applications, normalized cell indexis derived as normalized relative to the time point immediately before acompound addition or treatment. Thus, normalized cell index at such timepoint (immediately before compound addition) is always unit one for allwells. One possible benefit for using such normalized cell index is toremove the effect from difference in cell number in different wells. Awell having more cells may produce a larger impedance response followingcompound treatment. Using normalized cell index, it helps to remove suchvariations caused by different cell numbers.

In other embodiments, a cell change index (CCI) is calculated from thecell index. A “cell change index” at a given time point is calculated bysubtracting the cell index at a standard time point from the cell indexat the given time point. Thus, the cell change index is the absolutechange in the cell index from an initial time (the standard time point)to the measurement time. CCI is the normalized rate of change in cellindex. CCI values can be used to quantify the cell status change. Forcells in an exponential growth under regular cell culture condition, thecell index determined by a cell-substrate impedance monitoring systemdescribed herein is expected to be a proportionate measure of the cellnumber in the well since the cell morphology and average extent of celladhesion to the electrode surfaces among the whole cell population donot exhibit significant changes over time.

Turning back to comparing impedance based parameters or impedance basedcurves, in preferred embodiments, cell index is preferably calculatedand plotted over time to form an impedance base curve. Impedance basedcurves over time may be aligned or overlayed with one another accordingto electro-stimulation time points. For example, two or moreimpedance-based curves may be aligned or overlayed using a point or timeof electro-stimulation as a starting basis.

The skilled artisan will appreciate that when comparing impedance-basedparameters each member for comparison is a member of a same parameter.That is, impedance measurements can be compared to one another; cellindex, which can be calculated from the impedance measurement, can becompared to one another; or cell change index, which can be calculatedfrom cell index, can be compared to one another. Although individualmembers of a parameter can be compared, preferably the impedance basedparameters are plotted over time to provide an impedance-based curvethen compared, whether an impedance measurement curve, a cell indexcurve, or cell change index curve to identify differences changes inimpedance, which may be associated with administration of a compound,expression of an inserted nucleic acid or the like. In preferredembodiments, cell index is calculate from impedance measurements andplotted over time to provide an impedance-based curve in the form of acell index curve.

Extracellular recording measures the extracellular potential (alsoreferred to as field potential (FP)) and is a close simulation to itscounterpart, intracellular action potential (AP), therefore identifyingpeaks and wave form changes in field potential can be used to prediction channel activity. Accordingly, the extracellular recordingmeasurements are plotted over time, typically in microvolts, to identifyvariations in the field potential (FP). Sampling rate for extracellularrecording can be about 1 KHz, 2 KHz, 5 KHz and 10 KHz. Extracellularpotential is preferably plotted over time to form a field potentialcurve.

Periods or electro-stimulation intervals from two or more impedancecurves and/or two or more field potential curves may be overlayed toidentify trends or differences in impedance and/or field potential. Forclarity, the two or more impedance curves can be from different wells,such as from wells having serial dilutions of a compound or may be froma same well at a later time point. Accordingly, the addition ofcompounds suspected of affecting either the field potential (likely dueto affecting ion channels) or impedance of the excitable cells may beadded to a culture of cells and changes in impedance or field potentialcan be identified or measured through the comparison of the animpedance-based parameter and field potential before and after compoundaddition, such as by comparing cell index curves or field potentialcurves over time. Accordingly, in preferred embodiments comparisons areperformed by comparing cell index curves over time to one another andcomparing field potential curves over time to one another. In instanceswhere it is not readily apparent by viewing each curve over time, curvesbetween different electro-stimulation periods can be overlayed andcompared using curve comparison algorithms. Accordingly, overlayingimpedance curves may identify changes in impedance and overlaying fieldpotential curves may identify changes in field potential.

The methods are useful in assessing the cardiotoxicity of compoundsthrough monitoring impedance of a beating cell population andidentifying any changes after compound administration. For instance, acompound suspected of affecting excitation contraction coupling of theexcitable cells can be provided to the well. Impedance monitoring can beperformed before and after adding the compound and impedance-basedparameters (such as cell index) prior to and after adding the compoundcan be calculated and compared to identify changes in the impedanceparameter in response to the compound and thus predict whether thecompound is likely to be cardiotoxic. Preferably, the impedance basedparameter is compared between at least two different electro-stimulationintervals; however, three or more, four or more, five or more, six ormore and the like intervals can be compared.

In some embodiments, impedance and/or extracellular recording isconducted to assess an effect of a compound, such as its potentialcardiotoxicity, on cell. An exemplary method would be to provide aculture of cells, electro-stimulate the cells, perform impedancemonitoring and/or extracellular recording before and after adding thecompound and analyzing the results. As such, the analysis could includecalculating an impedance-based parameter (such as cell index or cellchange index) from impedance prior to and after adding the compound andtheir comparison to identify changes in the impedance parameter.Further, the impedance-based parameter may be plotted over time to forman impedance-based curve before and after administration for comparison.Alternatively or in addition, extracellular potential before and afteradministration can be compared such as by comparing field potentialcurves over time to identify differences. Such analysis in response tothe compound can be used to predict whether the compound is likely to becardiotoxic, affects beating, affects cardiomyocyte morphology oraffects ion channels. Preferably, the impedance based parameter iscompared between at least two different electro-stimulation intervals;however, three or more, four or more, five or more, six or more and thelike intervals can be compared. Compounds for testing are not intendedto be limiting and may include organic or inorganic molecules, drugs,peptides, proteins, antibodies, siRNA, shRNA, miRNA, cDNA, lipids or anycombination thereof.

While the above has been primarily discussed with respect to monitoringthe excitable cells before and after adding a compound suspected ofhaving a cardiotoxic affect to a well and comparison of impedance orfield potential before and after administration, in related approachesthe compound can be added to two or more wells in differentconcentrations to evaluate cardiotoxicity or activity compared to doseor can be added to two or more wells and administered in same well withan antagonist, such as to further verify suspected findings or mechanismof action. For instance, impedance-based curves can be used to calculatethe compound dose-dependent changes in cardiomyocyte morphology, ionchannel modulation, impedance, field potential or the like and generatean EC-50 value for the potency of the compound. In addition,extracellular recording permits comparisons with cells at variousdevelopmental stages to assess development.

Still further, analysis of impedance curves or extracellular recordingcurves after compound administration can be clustered and groupedaccording to similarities thereby being suggestive of a common mechanismof action. Further, once profiles of known compounds are grouped,unknown compounds can then be tested in such a system, and theimpedance-based curves and extracellular recording curves compared tothe group, then the compound can be assigned to one or more group basedon similarities or differences between curves.

In view of the above description and the examples that follow, it shouldbecome evident that while systems of the invention can be applied forcardiac electrophysiology recording for drug/compound profiling andcardiac safety screening, it can also be used to record from anyexcitable preparations. With incorporation of cell electro-stimulation,field potential recording and cell-impedance monitoring technologies,this system is the first single instrument which can simultaneouslymonitor the cardiac excitation by field potential and beatingfunctioning by impedance in vitro, yet the device still addselectro-stimulation of cells to induce or regulate excitationcontraction coupling or beating of a cell population and thus permitsuse of cells that require stimulation to begin or continue beating.

Assessing Genetically Manipulated Embryonic Stem Cells and Effects onCardiomyocyte Function

In another aspect, the present invention is directed to method to assessthe effect of gene knockout in an embryonic stem (ES) cell upondifferentiating into to a cardiomyocyte, the method including providinga device for monitoring impedance operably connected to an impedanceanalyzer, wherein the device includes at least two wells; addingwildtype ES cells as control to at least a first well and ES cells witha gene knockout in at least a second well; optionallyelectro-stimulating cells in each well; monitoring impedance of the atleast two wells and optionally determining cell indices from impedancevalues to generate an impedance-based curve for each well; comparing theimpedance-based curves between the first and second wells, and ifsignificantly different, concluding that the gene knockout may affect atleast one selected from the group consisting of cardiomyocyte viability,cardiomyocyte morphology, cardiomyocyte beating.

In a related approach, the invention also provides a method to assessthe effect of gene knockout in an embryonic stem (ES) cell upondifferentiating into to a cardiomyocyte, the method including: providinga device for monitoring impedance and extracellular recording, that areoperably connected to an impedance analyzer and extracellular recordingamplifier, wherein the device includes at least two wells; addingwildtype ES cells as control to at least a first well and ES cells witha gene knockout in at least a second well; monitoring impedance andextracellular recording of the at least two wells and optionallydetermining cell indices from impedance values to generate animpedance-based curve for each well; comparing the impedance-basedcurves and field potential curves between the first and second wells,and if significantly different, concluding that the gene knockout mayaffect at least one selected from the group consisting of cardiomyocyteviability, cardiomyocyte morphology, cardiomyocyte beating or affectingan electrophysiological property of the cardiomyoctye.

In another aspect, the present invention is directed to method to assessthe effect of a transgene in an embryonic stem (ES) cell upondifferentiating into to a cardiomyocyte, the method including providinga device for monitoring impedance operably connected to an impedanceanalyzer, wherein the device includes at least two wells; addingwildtype ES cells as control to at least a first well and ES cellsharboring a transgene knockout in at least a second well; optionallyelectro-stimulating cells in each well; monitoring impedance of the atleast two wells and optionally determining cell indices from impedancevalues to generate an impedance-based curve for each well; comparing theimpedance-based curves between the first and second wells, and ifsignificantly different, concluding that the transgene may affect atleast one selected from the group consisting of cardiomyocyte viability,cardiomyocyte morphology, cardiomyocyte beating.

In a related approach, the invention also provides a method to assessthe effect of a transgene in an embryonic stem (ES) cell upondifferentiating into to a cardiomyocyte, the method including: providinga device for monitoring impedance and extracellular recording, that areoperably connected to an impedance analyzer and extracellular recordingamplifier, wherein the device includes at least two wells; addingwildtype ES cells as control to at least a first well and ES cellsharboring a transgene in at least a second well; monitoring impedanceand extracellular recording of the at least two wells and optionallydetermining cell indices from impedance values to generate animpedance-based curve for each well; comparing the impedance-basedcurves and field potential curves between the first and second wells,and if significantly different, concluding that the transgene may affectat least one selected from the group consisting of cardiomyocyteviability, cardiomyocyte morphology, cardiomyocyte beating or affectingan electrophysiological property of the cardiomyoctye.

EXAMPLES Example 1 Specifications of an Exemplary System forElectro-Stimulation, Impedance Measurement and Extracellular Recording

A system was constructed for electro-stimulation of excitable cells, aswell as for impedance measurement and extracellular recording ofexcitable cells having two electrodes for extracellular recording, andshared electrodes in the form of a pair of interdigitated electrodestructures having a circle on line configuration that function both aselectro-stimulation electrodes an impedance monitoring electrodes. Thesystem was designed with the following specifications. The hardwaresystem bandwidth was 1 Hz˜2 KHz (−3 dB). The suppression ratio ofimpedance stimulation signal was >60 dB. The noise level was 10 μV(Vp-p). The system had excellent shielding and electromagneticcompatibility (EMC).

In regards to the electro-stimulation signal available waveforms arerectangular form, a ramp form, and sinusoidal. The polarity can be setfor uni-polarity or bi-polarity. An exemplary amplitude is−2.5V˜+2.5V@±100 mA per channel. An exemplary output voltage resolutionis 2 mV. An exemplary rise time (10 to 90%) of voltage: 0.2 μs @ ΔU=2V.An exemplary time lag between stimulation and voltage output is 5±1 μs @amplitude >200 mV. An exemplary current output is −250 mA˜+250 mA@perchannel. An exemplary resolution is 12 bit. An exemplary time resolutionis 10 μS. A maximum frequency (rectangular waveform) is 50 KHz. Astimulation format can stimulate all 96 wells in 96 well platesimultaneously or by column (8 wells). Further, in some instancesimpedance and extracellular recording measurement could be conductedsimultaneously with stimulation.

The sampling rate for impedance measurement over 96 wells was <10 ms.The sampling rate for extracellular recording signal was 1 KHz, 2 KHz, 5KHz, and 10 KHz. Maximal sampling channels for ECR signal was maximally8 channels (wells) in parallel, recorded from either one of twoelectrocellular recording electrodes in a single well.

Data acquisition mode permits extracellular recording only, impedancemeasurement only, and extracellular recording and impedance measurementsimultaneously.

A gain setting offers 1 K, 2 K, 4 K, and 8 K. ADC resolution is 14bit/±5V, total AD conversion rate of 800K SPS. Temperature rise in awell was lower than 0.4 C temperature rise compared to incubatortemperature. DC offset is fixed at 0.05 Hz, ±1 mV. Input voltage rangeis ±2.5 mV. Electrode input impedance of gold having a diameter 60μm/100 μm is <300 KΩ/1 KHz

Example 2 Monitoring the Spontaneous Field Potential Firing and Changesin Impedance of Cultured Cells

HL-1 atria cells were harvested and cultured in wells having a pair ofextracellular recording electrodes. FIG. 6A shows results from eightparalleled ECR signals in the HL-1 atria cells at day 7 with 40,000cells per well, some channels showed spontaneous field potential (FP)firing but no impedance (IMP) signals. As can be seen, the firing is notpaced at a regular rate.

CorAT cells (mouse embryonic stem cell derived cardiomyocytes) werecultured over time. At day 6, with a cell density at 45,000 per well,spontaneous field potentional (FP) firing was detected as well aschanges in impedance (IMP) as shown in FIG. 6B.

While the above experiments demonstrate the usefulness of ECR andimpedance monitoring of cells, spontaneous firing and impedance changeswere detected at different time points across different samples

Example 3 Modulation of Ion Channels while Performing ExtracellularRecording and Impedance Monitoring

CorAt cells were cultured over time and ECR and IMP signals wererecorded simultaneously. Different doses of Sotalol were added at day 9with CorAt cells at a density of 40,000 cells per well. FIGS. 7B-C showresults from 200 μM and 400 μM. FIG. 5A is control without Sotalol, FIG.5B shows treated wells or test wells with 200 μM, and FIG. 5C showstreated wells or test wells with 400 μM.

Administration of 400 μM sotalol induced a typical EAD and arrythmiaswhich indicating the primarily target of sotalol is the IKr (hERG) as itwas well characterized and published in literature. The other ionchannels (INa and ICa) were secondarily inhibited; this may due thedelay of recovery of membrane potential repolarization, so the availableportion of excitable INa and ICa channels were reduced.

Primary rat cardiomyocytes were cultured over time and ECR and IMPsignals were recorded simultaneously. Different doses of Quinidine wereadded at day 2 with the cardiomyocytes at 20,000 cells per well. FIG. 8Ashows an ECR and IMP overlay with Quinidine added at 0 μM. FIG. 8B showsan ECR and IMP overlay with Quinidine added at 2 μM. FIG. 8C shows anECR and IMP overlay with Quinidine added at 4 μM. FIG. 8D shows an ECRand IMP overlay with Quinidine added at 8 μM. FIG. 8E shows an ECR andIMP overlay with Quinidine added at 16 μM. FIG. 8F shows an ECR overlayof from FIGS. 8A-E. FIG. 8G shows an overlay of impedance-based curvesof FIGS. 8A-E.

Example 4 Field Stimulation and Pacing of Mouse Embryonic Stem CellDerived Cardiomyocytes (Cor.AT) Using the RTCA Cardio-ECR System

Cor.AT cells are mouse embryonic stem cell derived cardiomyocytes thatare engineered to express the GFP protein and puromycin protein forselection. These cells are a 100% pure population of cardiomyocytes thatbeat spontaneously in culture and express all the repertoire of ioniccurrents typical of a normal cardiomyocytes such as calcium, potassiumand sodium.

To assess the field stimulation and pacing of Cor.AT cells, the cellswere seeded in the wells of Cardio-ECR plates coated with fibronectin at10 ug/mL and at a density of 35,000 cells/well. This density has beenempirically optimized to sustain a cell monolayer that can beat in asynchronous manner. The cells were allowed to form a monolayer and themedia was changed to fresh media on daily basis. On the 9^(th) day afterseeding the cells were subjected to field stimulation. Thecardiomyocytes were field stimulated at 1.2 second duration with abiphasic pulse of 1 ms total length and an output voltage of 1.05 V. Thetotal duration of stimulation was for 40 seconds.

Both the field potential and impedance signals were dynamicallymonitored by the Cardio-ECR System. As shown in FIG. 9, application ofthe stimulation pulse caused regular pacing of the Cor.AT cells asmeasured primarily by the impedance signal (Red Trace) which signifiescontraction of the cardiomyocytes. The actual ECR signal during thestimulation phase (Blue Trace) is overwhelmed by the field stimulationsignal but can be observed when there is no stimulation.

Example 5 Field Stimulation and Pacing of Rat Neonatal CardiomyocytesUsing the RTCA Cardio-ECR System

Rat neonatal primary cardiomyocytes are spontaneously beatingcardiomyocytes that are used as a model system for studying heartfunction. This model is well-established for the study of the transportand toxicity of drugs and electrophysiological characterization.Neonatal rat cardiomyocytes are prepared and harvested as follows:

Briefly, hearts from neonatal rats are rapidly excised and washed toremove blood and debris. The ventricles are carefully minced anddissociated into single cells by proteolytic enzymes during repetitivedigestions with gentle stirring. Suspending cells in serum containinggrowth medium terminate the proteolytic digestion. Primarycardiomyocytes are purified and enriched by pre-plating twice for 45minutes each time. The cells are counted and adjusted to a desirableconcentration and then directly seeded in the wells of the Cardio-ECRPlates coated with 0.1% gelatin at a density of 14,000 cells/well. Thisdensity has been empirically optimized to sustain a cell monolayer thatcan beat in a synchronous manner. The cells were allowed to form amonolayer and the media was changed to fresh media on daily basis. Onthe 5^(th) day after seeding the cells were subjected to fieldstimulation. The cardiomyocytes were field stimulated at 0.8 secondduration with a biphasic pulse of 1.0 ms total length and an outputvoltage of 1.2V.

Both the field potential and impedance signals were dynamicallymonitored by the Cardio-ECR System. As shown in FIG. 10, application ofthe stimulation pulse caused regular pacing of the rat neonatal primarycells as measured primarily by the impedance signal (Red Trace) whichsignifies contraction of the cardiomyocytes. The actual ECR signalduring the stimulation phase (Blue Trace) is overwhelmed by the fieldstimulation signal but can be observed when there is no stimulation.

Example 6 Electro-Stimulation Coupled with Extracellular Recording ShowsChange in Field Potentials when Adding Different Concentrations of theCalcium Channel Blocker Isradipine

To show the benefit of coupling electro-stimulation with extracellularrecording, impedance and extracellular recording measurements wereperformed on HL-1 cells. Electro-stimulation was then applied atelectro-stimulation parameters of 1.1 V intensity, 1 ms pulse width, 1.3s stimulation interval. As shown in FIG. 11A, prior toelectro-stimulation, the HL-1 cells showed no measureable changes inimpedance (IMP) or extracellular recording field potential (FP);however, FIG. 11B shows electro-stimulation induced FP in theextracellular recording measurement and thus a “waking up” of the cells.

Cells were then treated with Isradipine, a L type Ca²⁺ channel blockerat either 0.25 μM (FIG. 11C), 0.5 μM (FIG. 11D), or 1 μM (FIG. HE). FIG.11F shows an overlay of the extracellular recording field potentials. Byapplication of the different doses of isradipine, the slow component ofthe induced FP (Ca channel) was gradually inhibited.

Example 7 Electro-Stimulation Coupled with Extracellular Recording ShowsChange in Field Potentials when Adding Different Concentrations of theCalcium Channel Activator Bay K8644 and Addition of Antagonist

As another example demonstrating the benefit of couplingelectro-stimulation with extracellular recording, impedance andextracellular recording measurements were performed on HL-1 cells. HL-1cells were cultured on a device having a pair of electro-stimulationelectrodes, a pair of impedance measurement electrodes and a recordingand reference electrode pair. Electro-stimulation was then applied atelectro-stimulation parameters of 1.1 V intensity, 1 ms pulse width, 1.3s stimulation interval. As shown in FIG. 12A, prior toelectro-stimulation, the HL-1 cells showed no measureable changes inimpedance (IMP) or extracellular recording field potential (FP);however, FIG. 12B shows electro-stimulation induced FP in theextracellular recording measurement and thus a “waking up” of the cellsfollowed by lack of FP after stopping electro-stimulation.

Cells were then treated with Bay K8644, a L type calcium channelactivator at 1 μM (FIG. 12C). The spontaneous firing rate was increaseddue to K8644 application, but its firing is irregular. The cells werethen electro-stimulation to pace the firing with the K8644 still in themedium using the electro-stimulation parameters of 1.1 V, 1 ms, 1.3 s asshown in FIG. 12D. The spontaneous firing rhythm was gradually adaptedto the stimulation frequency.

Cells were then treated with Nifedipine to antagonize the effect of BayK8644 at either 0.25 μM (FIG. 12E) or 0.5 μM (FIG. 12F). The stimulationinduced firing rate was remarkably inhibited due to nifedipineapplication. FIG. 12G shows the overlap of the field potential tracesfrom FIGS. 12A-F. Based on the overlapped trace analysis, we can seethat the FP was dose dependently inhibited by L type Ca2+ channelblocker, Nifedipine, the inhibition effect was mainly attributed to theblocking the Ca2+ peak (slow component followed by the initial fast Na+peak).

Example 8 Electro-Stimulation of CorAT Cells and the Pacing ofCardiomyocytes

CorAT cells were cultured on a device having a pair ofelectro-stimulation electrodes, a pair of impedance measurementelectrodes and a recording and reference electrode pair. At day 9 CorATcells with a density of 35,000 per well were paced by applying 1.05 V atevery 1.2 seconds for a duration of about 40 seconds. A shown in FIGS.13A-B, pacing was induced to produce reproducible plots of bothimpedance (shown in cell index) and extracellular field potential;however, when pacing by electro-stimulation stopped, both impedance andfield potential were irregular. In FIG. 13A, impedance and fieldpotential are overlayed whereas in FIG. 13B impedance is the upperdisplay and extracellular recording of field potential the lower.

FIG. 13C shows a single interval of electro-stimulation showing arelationship between change in impedance (top) and extracellularrecording field potential (lower)

Example 9 Electro-Stimulation of Rat Primary Cardiomyocytes and thePacing of Cardiomyocytes

Rat primary cardiomyocytes were harvested and cultured on a devicehaving a pair of electro-stimulation electrodes, a pair of impedancemeasurement electrodes and a recording and reference electrode pair. Atday 5, the cells had a cell density of 14K per well.

Cells were paced by applying electro-stimulation (1.2V, 1 ms, 0.8 s,stimulation duration: ˜40 s). FIGS. 14A-B showed the regular pacing ofthe cardiomyocytes and the reproducible impedance and field potentialresponses; however, when pacing stopped the regular impedance and fieldpotential profiles also stopped. FIG. 14A shows impedance as an overlaywith field potential while FIG. 14B shows impedance on the top panel andfield potential on the lower panel.

Example 10 Recording Neuronal Activity Through the Recording of FieldPotential

Our cardio ECR device includes a pair of extracellular recordingelectrodes, the size of which resemble that of popular multichannelelectrode recoding. We found that our electrode configurations could beused for recording of changes in field potential (FP) from culturedneuronal network (see example data below).

Dissociated neurons from brain or spinal tissue are seeded in the wellof our cardio ECR device, and allowed to mature over the course of threeweeks into self-organized networks complete with axons, dendrites andhundreds to thousands of synaptic connections. The neuronal networksexhibit spontaneous bursting activity after two weeks of incubation, andthe spontaneous activity could be monitored by the either one of the twoECR electrodes.

The neuronal firing data was recorded by either one of the two ECRelectrodes in single well. After performing measurements from the ECRelectrodes, Bicuculline, a GABAAR inhibitor, was added to the culture.As shown in FIG. 15A, a remarkable increased the FP spontaneous firingfrequency in cultured rat cortical neurons was measured on day three.This result is consistent with a previous publication that bathapplication of bicuculline caused large increases in spontaneous spikefiring frequency (Ma Y L, Weston S E, Whalley B J, Stephens G J. Thephytocannabinoid Delta(9)-tetrahydrocannabivarin modulates inhibitoryneurotransmission in the cerebellum. Br J. Pharmacol. 2008 May;154(1):204-15.

As a complementary experiment, Glutamate was added to the culture ratcortical neurons and the field potential monitored. As shown in FIG.15B, Glutamate, a non-selective GluR agonist remarkably increased the FPspontaneous firing amplitude and frequency in rat cultured corticalneurons on day ten.

1. A system for monitoring excitable cells comprising a devicecomprising at least one well, each well having a bottom comprising anonconductive substrate, wherein the substrate has a surface suitablefor attachment of excitable cells; a power source configured to deliveran electrical signal capable of electro-stimulating excitable cells; andat least one analyzing module for measuring an electrical property fromelectro-stimulated excitable cells, characterized in that each wellcomprises: a pair of electro-stimulation electrodes configured toreceive the electrical signal from the power source thereby deliveringan electro-stimulating signal to the well for electro-stimulation ofexcitable cells attached to the substrate; and at least a second pair ofelectrodes communicatively coupled to the at least one analyzing module,which is selected from the group consisting of a pair of impedancemonitoring electrodes communicatively coupled to the at least oneanalyzing module in the form of an impedance analyzer thereby permittingimpedance monitoring of excitable cells attached to the substrate, andan extracellular recording electrode pair communicatively coupled to theat least one analyzing module in the form of an extracellular recordingamplifier thereby permitting extracellular recording of excitable cellsattached to the substrate.
 2. The system according to claim 1, whereinthe electrical signal is a series of pulses at a regular time interval.3. The system according to claim 2, wherein the regular time interval isfrom 0.5 seconds to 2 seconds.
 4. The system according to claim 1,wherein the electrical signal is 1V to 2.5V for 0.5-2 milliseconds. 5.The system according to claim 1, wherein a percentage of a surface areaof the bottom of the at least one well occupied by the pair ofelectro-stimulation electrodes is selected from the group consisting of5% or more, 10% or more, 20% or more, 30% or more, 50% or more, and 70%or more.
 6. The system according to claim 1, wherein each of theelectro-stimulation electrodes independently comprises an unbranchedelectrode structure or a branched electrode structure.
 7. The systemaccording to claim 1, wherein the at least second pair of electrodes isthe pair of impedance monitoring electrodes and the analyzing module isin the form of the impedance analyzer.
 8. The system according to claim7, wherein the pair of impedance monitoring electrodes is a pair ofinterdigitated electrode structures, wherein each electrode structurecomprises a plurality of electrode elements.
 9. The system according toclaim 7, wherein the pair of impedance monitoring electrodes is a pairof electrode structures having a same surface area.
 10. The systemaccording to claim 7, the system comprising the extracellular recordingelectrode pair as a third pair of electrodes.
 11. The system accordingto claim 10, wherein at least one electrode of the pair ofelectro-stimulation electrodes is also at least one electrode of theextracellular recording electrode pair thereby permittingelectro-stimulation of excitable cells and extracellular recording ofattached cells using a same electrode at different time points.
 12. Thesystem according to claim 1, wherein the impedance analyzer monitorsimpedance at millisecond time resolution.
 13. The system according toclaim 1, wherein the at least second pair of electrodes is theextracellular recording electrode pair and the analyzing module is inthe form of the extracellular recording amplifier.
 14. The systemaccording to claim 13, wherein the extracellular recording electrodepair comprises a recording electrode and a reference electrode, whereinthe recording electrode has a diameter from about 10 μm to about 200 μmor from about 30 μm to about 100 μm.
 15. The system according to claim13, wherein the extracellular recording electrode pair comprises arecording electrode and a reference electrode, further wherein a ratioof the area of reference electrode to the area of recording electrode isselected from the group consisting of 2 or more, 10 or more, 100 ormore, 1,000 or more, and 10,000 or more.
 16. The system according toclaim 13, further comprising a second recording electrode having a samediameter as a first.
 17. The system according to claim 13, wherein atleast one electrode of the pair of electro-stimulation electrodes isalso at least one electrode of the pair of impedance monitoringelectrodes thereby permitting electro-stimulation of excitable cells andimpedance monitoring of attached cells using a same electrode atdifferent time points.
 18. The system according to claim 17, wherein thepair of impedance monitoring electrodes is a pair of interdigitatedelectrode structures, wherein each electrode structure comprises aplurality of electrode elements.
 19. The system according to claim 18,wherein at least one electrode of the pair of impedance monitoringelectrodes is the reference electrode.
 20. A device for monitoringexcitable cells, the device comprising: a) at least one well, each wellhaving a bottom comprising a nonconductive substrate, wherein thesubstrate has a surface suitable for attachment of excitable cells; b) apair of electro-stimulation electrodes positioned on the substratewithin the at least one well and configured to electro-stimulate theexcitable cells; and c) at least a second pair of electrodes positionedwithin the at least one well and selected from the group consisting of apair of impedance monitoring electrodes and extracellular recordingelectrode pair, wherein the pair of impedance monitoring electrodes areconfigured for monitoring cell-substrate impedance of cells attached tothe substrate, and wherein the extracellular recording electrode pairare configured for monitoring extracellular potential of cells attachedto the substrate. 21-36. (canceled)
 37. A method for monitoringexcitable cells, comprising: a) providing the system according to claim1; b) adding a sample of excitable cells to the device; c)electro-stimulating the excitable cells; and d) monitoringelectro-stimulated cells through the at least second pair of electrodes.38-61. (canceled)